Simultaneous detection of cannibalism and senescence as prognostic marker for cancer

ABSTRACT

The present inventors show that cannibal cells can undergo senescence after entosis in vivo and that the tumor suppressive protein p53 act as a repressor of this phenomenon. They therefore propose new tools to study the molecular pathways involved in the cannibalism process, for example by measuring the expression levels of p53 or splice variants thereof (such as Δ133TP53, TP53β, TP53γ or Δ40TP53), the release of extracellular ATP or purinergic P2Y2 receptor activity. The present inventors also demonstrated that the detection of senescent cannibal cells in breast adenocarcinoma obtained from patients treated with neo-adjuvant therapy positively correlates with good patient&#39;s response to treatment. Altogether, these results provide the first evidence that detection of cellular cannibalism and senescence simultaneously in tumors helps for the diagnosis of disease outcomes and for the prediction of treatment efficiency against cancer diseases.

This application is a divisional of U.S. patent application Ser. No.14/412,571, filed Jan. 2, 2015, now U.S. Pat. No. 9,726,661, which is aU.S. National Stage of PCT/EP2013/064408, filed Jul. 8, 2013, whichclaims priority to U.S. Provisional Application No. 61/668,775, filedJul. 6, 2012, all of which are incorporated herein in their entirety byreference.

SUMMARY OF THE INVENTION

The present inventors show that cannibal cells can undergo senescenceafter entosis in vivo and that the tumor suppressive protein p53 act asa repressor of this phenomenon. They therefore propose new tools tostudy the molecular pathways involved in the cannibalism process, forexample by measuring the expression levels of p53 or splice variantsthereof (such as Δ133TP53, TP53β, TP53γ or Δ40TP53), the release ofextracellular ATP or purinergic P2Y2 receptor activity. The presentinventors also demonstrated that the detection of senescent cannibalcells in breast adenocarcinoma obtained from patients treated withneo-adjuvant therapy positively correlates with good patient's responseto treatment. Altogether, these results provide the first evidence thatdetection of cellular cannibalism and senescence simultaneously intumors helps for the diagnosis of disease outcomes and for theprediction of treatment efficiency against cancer diseases.

BACKGROUND OF THE INVENTION

Cannibalism constitutes a consumption strategy used by micro- and higherorganisms to adapt to environmental stresses and to survive. Grampositive species, Bacillus subtilis and Streptococcus pneumonia exertcannibalistic activities during the early stages of sporulation(Gonzalez-Pastor et al., Science 2003) or during natural genetictransformation (Guiral et al., PNAS 2005). In contrast, the slime moldeDictyostelium caveatum represses its predatory abilities during itsquasi-multicellular differentiation stage (Waddell and Duffy, TheJournal of cell biology 1986). In Drosophila, studies on genetic mosaicsthat place cells in competition within tissues unrevealed thatcannibalism is a genetically controlled process that may occur at thesingle cell level and actively participates to cell competition duringtissue repair and tumor development (Li and Baker, Cell 2007). Althoughcellular cannibalism is poorly reported in physiological situations,cannibal cells have been frequently detected in various human tumortypes such as melanoma, leukemia and cervical carcinoma, coloncarcinoma, stomach carcinoma, liver carcinoma adenocarcinoma and inmetastatic breast carcinoma (Overholtzer and Brugge, Nature reviewsMolecular cell biology 2008).

On a one hand, cellular cannibalism was suggested to cause thedestruction of cancer cells by other malignant cells by entosis. Incontrast to oilier (apoptotic, necrotic or autophagic) cell death formsthat are controlled in a cell-autonomous fashion, entosis (from theGreek word entos, which means inside, into, or within) requires theinternalization of a live target cancer cell by a live <<cannibal >>cancer cell (Overholtzer and Brugge, Nature reviews Molecular cellbiology 2008). An inverse correlation between entosis and metastasisappearance in human pancreatic adenocarcinoma was furthermore reported,suggesting that this atypical death process may represent intrinsictumor suppression mechanism (Cana et al., EMBO Mol Med. 2012).

On another hand, cellular cannibalism was shown to provoke thepolyploidization of the engulfing cell by disrupting cytokinesis, andhence to promote oncogenesis indirectly, by generating polyploid cellsthat tend to generate aneuploidy daughter cells (Krajcovic et al., Nat.Cell Biol. 2011; Krajcovic and Overholtzer, Cancer Res. 2012).

As a consequence of (homotypic or heterotypic) interactions betweencancer cells or between cancer cells and other (stromal or immune)cells, cannibalism could have—depending on the genetic status of cancercells (target and cannibal cells) and on tumor microenvironment—variableconsequences on tumor growth and disease outcomes.

Although the “cell-in-cell” cytological features have been widelyreported in human tumors, the molecular and cellular bases of cellularcannibalism remain unknown.

In this context, the present inventors provide evidence that TP53 and,more specifically, Δ133TP53, acts as repressors of cellular cannibalism.Their results also unrevealed that cellular cannibalism is functionallyconnected to senescence, both in vitro (in cellular models ofsenescence) and in vivo (in human breast carcinoma), where they mayinfluence the efficiency of a chemotherapeutic treatment.

FIGURE LEGENDS

FIG. 1. Identification of TP53 as repressor of cellular cannibalism. (A)Schematic representation of experimental procedure used to evaluate theability to tumor suppressive depletion to enhance cell in cellinternalization. (B) Frequencies of cell-in-cell structures induced bydepletion of tumor suppressive proteins in HCT116. After 48 hours oftransfection indicated siRNA, colorectal HCT116 cells were stained withCMTMR and CMFDA cell trachers, mixed and cultured during 24 hours.Quantification of cell-in-cell structures induced by depletions of tumorsuppressor proteins was realized by confocal microscopy. The frequenciesof cell-in-cell structures were determined for at least 300 cells in 3independent experiments (mean+/−s.e.m, p<0.001). (C) Detection ofcell-in-cell structures induced by depletion of TP53, as visualized byconfocal microscopy. Inserts represent xz and yz optical sections andshow that after p53 depletion, red CMTR labeled HCT116 cells areinternalized by green CMFDA labeled HCT116 cells. Representativemicrographs of xy (scale bar, 5 μm), xz (scale bar, 4 μm) and yz (scalebar, 1 μm) optical sections are shown. Images are representative of atleast four independent experiments. (D) Representative micrographs ofcell-in-cell structures showing that after depletion of TP53 by twospecific siRNAs, (red) CMTR labeled HCT116 cells internalized (red) CMTRlabeled HCT116 cells (noted R(R)) or (green) CMFDA labeled HCT116 cells(noted R(G)), and (green) CMFDA labeled HCT116 cells engulfed (red) CMTRlabeled HCT116 cells (noted G(R)) or (green) CMFDA labeled HCT116 cells(noted G(G)) (scale, 5 μm). (E) Frequencies of cell-in-cell structuresshowing R(R), R(G), G(R) or G(G) cell internalization detected afterdepletion of TP53 by two distinct siRNAs (as compare to control). Thefrequencies of cell-in-cell structures were determined for at least 300cells in 3 independent experiments (mean+/−s.e.m, p<0.001). (F)Cell-in-cell structures induced by 10 μM PFT-α, TP53 knockdown, or TP53knockout in HCT116. Cell-in-cell structures were identified by b-catenin(white), CMFDA (green) and CMTMR (red) staining of internalizing HCT116cells. Images are representative of at least four independentexperiments (scale bar in a, 5 μm and scale bar in b 1 μm). (G)Frequencies of cell-in-cell structures induced by 10 μM PFT-α, TP53knockdown, or TP53 knockout in HCT116 in presence or in absence of 20 μMof ROCK inhibitor (Y27632) or 100 μM of pan-caspase inhibitor (z-VAD).The frequencies of cell-in-cell structures were determined for at least300 cells in 3 independent experiments (mean+/−s.e.m, p<0.001). (H)Detection of cell-in-cell structures in TP53 depleted human diploidfibroblasts strain WI38, as visualized by confocal microscopy. Insertsrepresent xz and yz optical sections and show that after TP53 depletion,red CMTR labeled primary WI38 cells are internalized by green CMFDAlabeled HCT116 cells. Representative micrographs of xy (scale bar, 5μm), xz (scale bar, 4 μm) and yz (scale bar, 1 μm) optical sections areshown. Images are representative of at least four independentexperiments. (1) Frequencies of cell-in-cell structures induced bydepletion of TP53 in human diploid fibroblasts strain WI38. Thefrequencies of cell-in-cell structures were determined for at least 300cells in 3 independent experiments (mean+/−s.e.m, p<0.001).

FIG. 2. Δ133TP53 isoform is also a repressor of cellular cannibalism.(A-D) Effects of TP53 mutants and TP53 isoforms on cell-in-cellinternalization in Tp53^(−/−) HCT116 cells. Schematic representation ofTP53 mutants and isoforms used in this study (A). Expression of TP53mutants in Tp53^(−/−) HCT116 cells was validated by Western blot.Immunoblot shown is representative of 3 independent experiments (B).Representative micrographs of Tp53^(−/−) transfected with plasmidsexpressing WI (TP53^(WT)), nuclear (TP53^(NES−)), cytoplasmic(TP53^(NLS−)) or mutated TP53 (TP53^(R175H) and TP53^(R273H)) are shown(C). Quantification of cell-in-cell structures of Tp 53^(−/−) cellstransiently transfected with TP53 mutants is shown (mean+/−s.e.m, n=3,p<0.001) (D). Micrographs of Tp53^(−/−) cells transfected with plasmidsexpressing wild type TP53, TP53β, TP53γ, Δ40TP53 or Δ133TP53 isoformsare shown (E). Quantification of cell-in-cell structures of Tp53^(−/−)cells transiently transfected with wild type TP53 or TP53 isoforms(TP53, TP53β, TP53γ, Δ40TP53 or Δ133TP53) is shown (mean+/−s.e.m, n=3,p<0.001) (F). (G-I) Effects of Δ133TP53 depletion on cell-in-cellinternalization in HCT116 cells. Depletion of Δ133TP53 in HCT116 cellswas validated by Western blot (n=3)(G). Detection of cell-in-cellstructures induced by depletion of Δ133TP53 by confocal microscopy.Representative micrographs of xy (scale bar, 5 μm), is shown. Image isrepresentative of at least four independent experiments (K). Frequenciesof cell-in-cell structures induced by depiction of Δ133TP53 in humandiploid fibroblasts strain WI38. The frequencies of cell-in-cellstructures were determined for at least 300 cells in 3 independentexperiments (mean+/−s.e.m, p<0.001) (L). Effects of TP53 or Δ133TP53knockdown and TP53 knock out on cell engulfment. Quantification ofcell-in-cell structures obtained after culture of TP53- orΔ133TP53-depleted HCT116 cells (red) with CMFDA labeled HCT116 targetcells is shown (mean+/−s.e.m, n=3, p<0.001) (M).

FIG. 3. Extracellular ATP and purinergic receptor P2Y2 participate incellular cannibalism induced by TP53 or Δ133TP53 depletion. (A) Releaseof ATP during coculture of Tp53^(−/−) (black circle) or Tp53^(+/+)(white circle) HCT116 cells was determined at different time points byATP-dependent bioluminescence in 3 independent experiments. Onerepresentative experiment is shown (mean±SEM of triplicates; *P <0.01).(B) Effect of apyrase on cellular cannibalism. Cocultures of TP53 orΔ133TP53 depleted HCT116 cells in presence of different concentrationsof apyrase were performed. Then, detection of cell-in-cell structureswas performed by confocal microscopy (mean+/−s.e.m, n=3, p<0.001). (C,D)Effects of extracellular ATP and UTP on cellular cannibalism. Detectionof cell-in-cell structures induced after the supplementation of ATP orUTP on culture of CMFDA and CMTMR labeled HCT116 cells. Representativemicrographs are shown (scale bar, 5 μm). Images are representative of atleast 3 independent experiments (C). Frequencies of cell-in-cellstructures induced by ATP or UTP supplementations in HCT116 in presenceor in absence of 20 μM of ROCK inhibitor (Y27632) or of 100 μMpan-caspase inhibitor (zVAD). The frequencies of cell-in-cell structureswere determined for at least 300 cells in 3 independent experiments(mean+/−s.e.m, p<0.0011) (D). (E-F) Overexpression of P2Y2 after TP53 orΔ133TP53 depletions on colorectal carcinoma HCT116 cells. Expression wasdetermined by immunoblot. Representative immunoblots of 3 independentexperiments are shown. (G,H) Effects of P2Y2 depletion on cellularcannibalism observed after TP53 or Δ133TP53 depletions. As previouslydescribed, HCT116 (G) or PANC813 (H) cells that are depleted for P2Y2and/or inactivated for TP53 or Δ133TP53 were stained, mixed and culturedduring 24 hours in presence or in absence of 20 μM of Y27632 or 100 μMof zVAD. The frequencies of cell-in-cell structures were determined forat least 300 cells in 3 independent experiments (mean+/−s.e.m,*p<0.001). (I-J) P2Y2 expression on human primary fibroblasts after TP53or Δ133TP53 depletions. Expression was determined by immunoblot.Representative immunoblots of 3 independent experiments are shown. (K)Effects of pharmacological inhibition of P2Y2 on cellular cannibalisminduced by Δ133TP53 depletion. Human primary fibroblasts that aredepleted for Δ133TP53 were stained, mixed and cultured during 24 hoursin presence or in absence of the 20 μM of P2Y? inhibitor Kaempferol. Thefrequencies of cell-in-cell structures were determined for at least 300cells in 3 independent experiments (mean+/−s.e.m, p<0.001).

FIG. 4. Cellular cannibalism leads to senescence. (A,B) Effects ofcellular cannibalism on cell proliferation. After cell sorting, cellproliferation of TP53 or Δ133TP53 knocked down and TP53 knocked outsingle or cannibal cells were performed during 6 days. (C-H) Effects ofcellular cannibalism on senescence induction. Cannibal cells mediated byTP53 knockout or Δ133TP53 knockdown were examined in EdU incorporationassay (C,D), in DNA damage response (E,F) and in SA-β-Gal assay (G,H).(C) Determination of EdU incorporation on cannibal cells induced byΔ133TP53 knockdown, or TP53 knockout. Cell-in-cell structures wereidentified by β-catenin (red) and Hoechst 33342 (blue) staining ofinternalizing HCT116 cells. EdU positive cells are green. Images arerepresentative of at least three independent experiments (scale bar: 5μm). (D) Determination of EdU incorporation in single and cannibal cellsafter TP53 or Δ133TP53 inactivation. The number of EdU positive singleor cannibal cells per total number of cells examined (at least 300 perwell) was recorded. (E) Detection of DNA damage response (DDR) foci insingle and cannibal cells after TP53 or Δ133TP53 inactivation. DNAdamage response (DDR) foci were identified by γ-H2AX or p53BP1 (White),CMFDA (green), CMTMR (red) and Hoechst 33342 (blue) staining ofinternalizing HCT116 cells. Images are representative of at least threeindependent experiments (scale bar: 5 μm). (F) Quantification of DNAdamage foci in single and cannibal cells after TP53 or Δ133TP53inactivation. The percentage of γ-H2AX⁺ cells or p53BP1⁺ cells weredetermined by confocal microscopy. Error bars represent means±SEM (n=3;*P 0.01). Representative pictures of SA-β Gal staining of single andcannibal cells obtained after TP53 or Δ133TP53 inactivation. (H) Summaryof SA-β Gal assay. The data are mean±SEM (n=3; *P <0.01). (I) p21^(WAF1)expression during coculture of Tp53^(+/+) or TP53^(−/−) cells,P21^(WAF1) expression was determined by immunoblot. Representativeimmunoblots of 3 independent experiments are shown. (J) Detection ofp21^(WAF1) expression in cannibal cells after Δ133TP53 depletion.Expression of p21^(WAF1) was examined by confocal microscopy usingantibodies against p21^(WAF1) (white) or β-catenin (red). Nuclei arestained with Hoechst 33342 (blue). Images are representative of at leastthree independent experiments (scale bar: 5 μm). (K) Quantification ofp21^(WAF1) expression in single and cannibal cells after TP53 orΔ133TP53 inactivation. The percentage of □21WAF⁺ cells in cannibal cellswas determined by confocal microscopy. Error bars represent means±SEM(n=3; *P <0.01). (L) Expression of p21^(WAF1) on HCT116 Tp53^(−/−) afterknockdown of p21^(WAF1). P21^(WAF1) expression was determined byimmunoblot. Representative immunoblots of 3 independent experiments areshown. (M) Effects of p21^(WAF1) depletion on SA-βGal activity ofcannibal cells. The number of SA-β Gal positive single or cannibal cellsper total number of cells examined (at least 300 per well) was recorded.Error bars represent means±SEM (n=3; *P <0.01). (N) Effects ofp21^(WAF1) knockdown on SA-β Gal activity of cannibal cells obtainedafter TP53 or Δ133TP53 inactivation. Quantification of SA-β Gal positivecells were realized as described above (n=3; means±SEM; *P <0.01). (O)Effects of p21^(WAF1) knockdown on cell-in-cell internalization detectedafter TP53 or Δ133TP53 inactivation. Cell-in-cell structures wereidentified by β-catenin (white), CMFDA (green) and CMTMR (red) stainingof transfected HCT116 cells. The frequencies of cell-in-cell structureswere determined for at least 300 cells in 3 independent experiments(mean+/−s.e.m, p<0.001).

FIG. 5. Senescent cells exert cannibalistic activity. (A) Detection ofP2Y2 overexpression during oncogenic induced senescence. Expression ofP2Y2, p21^(WAF1), p16^(INK4b) and Rb during retroviral transduction ofoncogenic Ras^(V12) in human primary fibroblasts (W138) were determinedby immunoblot. Representative immunoblot of three independentexperiments was shown. GAPDH was used as loading control. (B) Detectionof cell-in-cell structures during coculture of human primary fibroblastsor RasV12 expressing human primary fibroblasts with human primaryfibroblasts, with RasV12 expressing human primary fibroblasts or withcolorectal HCT116 cells. Cell-in-cell structures were analyzed byconfocal microscopy. Inserts represent xz and yz optical sections andshow that Ras^(V12) expressing fibroblasts internalized neighboringcells (human primary fibroblasts or HCT116 cells—. Representativemicrographs of xy (scale bar, 5 μm) xz (scale bar, 4 μm) and yz (scalebar, 1 μm) optical sections are shown. Images are representative of atleast three independent experiments. (C) Frequencies of cell-in-cellstructures induced after transduction of oncogenic Ras^(V12) in humandiploid fibroblasts (W138). The frequencies of cell-in-cell structureswere determined for at least 300 cells in 3 independent experiments(mean+/−s.e.m, p<0.001). (D) Detection of P2Y2 overexpression duringreplicative stress induced senescence. Expression of P2Y2, p21^(WAF1),p16^(INK4b) and Rb during replicative senescence in human primaryfibroblasts (WI38) were determined by immunoblot. Representativeimmunoblot of three independent experiments was shown. GAPDH was used asloading control, (E) Frequencies of cell-in-cell structures inducedduring replicative stress in human diploid fibroblasts (WI38). Thefrequencies of cell-in-cell structures were determined for at least 300cells in 3 independent experiments (mean+/−s.e.m, p<0.001). (F) Effectsof pharmacological inhibition of P2Y2 on cellular cannibalism detectedduring replicative induced senescence. Coculture of replicative stressinduced senescent human fibroblasts with human primary fibroblast orcolorectal HCT116 cells were realized during 24 hours in presence or inabsence of 33 μM of Kaempferol. Then, frequencies of cell-in-cellstructures induced during replicative stress in human diploidfibroblasts (WI38) were determined for at least 300 cells in 3independent experiments (mean/−s.e.m, p<0.001). (G) Detection of P2Y2overexpression during replicative stress induced senescence on humanprimary keratinocyte. Expression of P2Y2, p16^(INK4b) and Rb duringreplicative senescence in human primary keratinocytes (HEKn) weredetermined by immunoblot. Representative immunoblot of three independentexperiments was shown. GAPDH was used as loading control. (H) Detectionof cell-in-cell structures during culture of human primary keratinocytesafter 3 passages (P3), 4 passages (P4) or 5 passages (P5). A previouslydescribed, cell-in-cell structures were analyzed by confocal microscopy.Inserts represent xz and yz optical sections and show that replicativestress induced senescent fibroblasts internalized neighboring cells(human primary fibroblasts or HCT116 cells). Representative micrographsof xy (scale bar, 5 μm), xz (scale bar, 4 μm) and yz (scale bar, 1 μm)optical sections are shown. Images are representative of at least threeindependent experiments. (I) Effects of pharmacological P2Y2 inhibitorof P2Y2 on cellular cannibalism detected during replicative inducedsenescence. Coculture of replicative stress induced senescent humanfibroblasts with human primary fibroblast or colorectal HCT116 cellswere realized during 24 hours in presence or in absence of 33 μM ofKaempferol, 20 μM of Y27632 and 100 μM of ZVAD. Then, frequencies ofcell-in-cell structures induced during replicative stress in humanprimary keratinocytes (HeKn) were determined for at least 300 cells in 3independent experiments (mean+/−s.e.m, p<0.001).

FIG. 6. Detection of cell-in-cell structures during culture of cancercell lines and validation of tumor suppressor knockdowns, (A)Frequencies of cell-in-cell structures detected during culture ofdifferent cell lines as previously described. HCT116, HT29, H1299,PANC813, H1975, H1650, A549, HCC1937, SQ20B, HeLA and U2OS cells warestained with CMTMR or CMFDA cell trackers, mixed and cultured during 24hours. Quantification of cell-in-cell structures was realized byconfocal microscopy. The frequencies of cell-in-cell structures weredetermined for at least 300 cells in 3 independent experimentsmean+/−s.e.m, p<0.001). (B-Q) Validation of tumor suppressor knockdownsafter transfection of two siRNA for Par-4 (B), BRCA-2 (C), PTEN (D), ATR(F), FOXO-1 (F), BRCA-1 (G), Beclin-1 (H), ATM (I), FOXO-4 (J), FOXO-3(K), Rb (L), TP63 (M), TP73 (N), LKB1 (O), p15^(INKb) (P) and TP53 (Q)was performed. Expression of indicated tumor suppressive proteins wasdetermined by immunoblot. Representative immunoblots of threeindependent experiments are shown. GAPDH was used as loading control.

FIG. 7. Effects of TP53 inactivation on cellular cannibalism. (A)Detection of cell-in-cell structures induced by depletion of TP53, asvisualized by confocal microscopy. Inserts represent xz and yz opticalsections and show that after p53 depletion, red CMTR labeled HCT116cells are internalized by green CMFDA labeled HCT116 cells. Images arerepresentative of at least four independent experiments. (B) Detectionof cell-in-cell structures induced by depletion of TP53 by lightmicroscopy. (C) Time-lapse imaging of cellular cannibalism detectedafter depletion of TP53 on HCT116 cells. After 48 hours of transfectionwith siRNA against TP53, cells were stained as previously described withCMFDA and CMTMR cell trackers and cocultured under time-lapse confocalvideomicroscopy. Indicated numbers represent hours and minutes.Time-lapse videomicroscopy image sequences shown are representative ofat least three independent experiments. (D) Absence of apoptoticcleavage of caspase-3 during depletion of TP53. After 48 hours of TP53knockdown, cleavage of caspase-3 was analyzed by immunoblot. Note that100 mM of cisplatin (COOP) used as positive control revealed intensecleavage of caspase-3. GAPDH was used as loading control. Representativeimmunoblots of three independent experiments were shown. (E) Analyze of20 independent Time-lapse videomicroscopy image sequences. Mitoticevents and cellular cannibalism were detected and recorded duringapproximately fifteen hours. (F) Impact of TP53 inactivation on ROCKactivity. ROCK activity was determined by detecting the phosphorylationof myosin light chain 2 on serine 19 (MLC2S19*). Lysates for 10 μM PFT-αtreated—. TP53 knocked down, or TP53 knocked out HCT116 cells wereanalyzed for MLC2S19*, MLC2 or TP53 by immunoblot. GAPDH was used asloading control. Representative immunoblots of three independentexperiments are shown, (G) Electron microscopy micrographies of cannibalcells. After 24 hours of culture. Tp53^(−/+) and Tp53^(−/−) HCT116 cellsare fixed and analyzed by electron microscopy. Representative electronmicroscopy micrographies of three independent experiments are shown.(H,I) Effects of TP53 depletion on TP53 expression and on MLC2S19*.HCT116 cells were transfected with specific siRNAs for TP53 during 48hours and expression of TP53 (H) or MLCS19* (I) were determined byimmunoblots. GAPDH was used as loading control. Representativeimmunoblots of three independent experiments are shown.

FIG. 8. Depletion of Δ133TP53 triggers cellular cannibalism of PANC813cells and of human primary fibroblasts. (A) Detection of cell-in-cellstructures after depletion of Δ133TP53 in PANC813 cells. After 48 hoursof transfection with two specific siRNAs for Δ133TP53, PANC813 cellswere stained as previously described, with CMFDA and CMTMR cell trackersand cocultured during 24 hours. Then, cells were fixed and stained forβ-catenin (white) and nuclei (blue). Detection of cell-in-cellstructures induced by depletion of Δ133TP53, as visualized by confocalmicroscopy. Representative micrographs are shown. Images arerepresentative of at least four independent experiments (scale bar, 5μm). (B) Analysis of Δ133TP53 depletion in human diploid fibroblasts.Expression of Δ133TP53 was GAPDH was used as loading control.Representative immunoblots of three independent experiments are shown.

FIG. 9. Internalized cells are degraded by cannibal cells. (A) Detectionof subcellular localization of lysosomal marker LAMP2 in cannibal cellsobtained after PFT-α treatment, TP53 knockdown, or TP53 knockout.Subcellular localization of LAMP2 on cannibal cells was examined byconfocal microscopy by LAMP2 (white), CMFDA (green), CMTMR (red) andHoechst 33342 (blue) staining of HCT116 (A) or PANC0813 (B) cells. (C)Quantification of LAMP2 recruitment to internalized cells. Thefrequencies of LAMP2 encircled nuclei were determined for at least 300cells in 3 independent experiments (mean+/−s.e.m, p<0.001). (D)Detection of lysosomal compartment in cannibal cells obtained aftercoculture of Tp53^(−/−) HCT116 cells. As in (A), Subcellularlocalization of lysosomal compartment on cannibal cells was examined byconfocal microscopy by β-catenin (white), LAMP2 (green), Lysotracker(red) and Hoechst 33342 (blue) staining of Tp53^(−/−) HCT116 cells. (E)Detection of target cell degradation during cellular cannibalism.Isogenic Tp53^(−/−) HCT116 cells that stably expressed green or redfluorescent proteins in their nuclei (GFP-histone H2B and RFP-histoneH2B fusion proteins were mixed and cocultured for 24 hours. Afterfixation, these cells were analyzed by confocal microscopy or byelectron microscopy and revealed that internalized cells are degraded.(F) Detection of nuclear degradation of internalized cells duringcellular cannibalism obtained after culture of isogenic Tp53^(−/−)HCT116 cells. Cannibal cells were examined by detecting EdU (green),α-tubulin (red), Hoechst 33342 (blue) stainings and phase contrast(grey) using confocal microscopy. Representative micrographs are shown.Images are representative of at least four independent experiments(scale bar, 5 μm), (G) Quantification of nuclear degradation ofinternalized cells during cellular cannibalism induced by TP53 andΔ133TP53 inactivation. The frequency nuclear degradation of internalizedcells was determined for at least 300 cells in 3 independent experiments(mean+/−s.e.m, p<0.001),

FIG. 10. Identification and characterization of cannibal cells aftercell sorting. Isogenic pairs of cancer cell lines (HCT116 Tp53^(+/−)PANC813) that stably expressed green or red fluorescent proteins intheir nuclei (GFP-histone H2B and RFP-histone H2B fusion proteins) weremixed, cocultured, optionally after depletion of Δ133TP53 to induceentosis, and seeded in microliter plates (with one single structure perwell) after cell sorting and analyzed by fluorescent microscopy. (A)Representative micrographs of cannibal cells obtained from HCT116 orPANC (Day 1 and Day 7) are shown. (B) Time-lapse videomicroscopy imagesequences shown are representative of at least three independentexperiments obtained after Δ133TP53 depletion,

FIG. 11. Extracellular ATP and purinergic receptor P2Y2 participate incellular cannibalism. (A.) Detection of cellular cannibalism aftercis-platinium (CDDP) or γ-irradiation (IR) treatment. After 24 hourstreatment with 10 μM CDDP or irradiation (IR) with 4 grays, half cellpopulation of colon carcinoma HCT116 cells were stained with green CMFDAcell tracker or with red CMTMR cell tracker and analyzed for cellularcannibalism by confocal microscopy after 24 hours of coculture inpresence or in absence of 20 μM of ROCK inhibitor (YI27632) or 100 μM ofpan-caspase inhibitor (zVAD). The frequencies of cannibal cells weredetermined for at least 300 cells in 3 independent experiments(mean+/−s.e.m, *p<0.001). (B-D) Detection of ATP release afterstimulation with senescent-induced stresses. Release of ATP on HCT116cells treated with 10 μM CDDP (B) or 4 gray γ-irradiation (IR) (B) Ofdepleted for p53 (C) or Δ133p53 (C) was determined 2 hours after thebeginning of coculture using ATP-dependent bioluminescence in 3independent experiments, (D) Effect of ROCK inhibitor on ATP releasedetected after p53 inactivation. Release of ATP was detected aspreviously described in presence or in absence of 20 μM of Y27632(mean+/−s.e.m, *p<0.001). (E) Effects of extracellular ATP and UTP oncellular cannibalism. Detection of cell-in-cell structures induced afterthe supplementation of ATP or UTP on culture of CMFDA and CMTMR labeledHCT116 cells. Representative micrographs are shown (scale bar, 5 μm).Images are representative of at least 3 independent experiments. Then,frequencies of cell-in-cell structures induced by ATP or UTPsupplementations in HCT116 in presence or in absence of 20 μM of ROCKinhibitor (Y27632), of 100 μM pan-caspase inhibitor (zVAD) or 20 μM. ofP2Y2 inhibitor Kaempferol. The frequencies of cell-in-cell structureswere determined for at least 300 cells in 3 independent experiments(mean+/−s.e.m, *p<0.001). (E-M) Analysis of purinergic receptor P2Y2expression after senescent-induced stresses. P2Y2 expression wasdetermined by immunoblot on WI38 cells after hypoxia (F), during cultureof human primary keratinocytes (HEKn) after 3 passages (P3), 4 passages(P4) or 5 passages (P5) (G), after irradiation with indicated doses ofhuman primary keratinocytes (HEKn) (H), after treatment of HCT116 cellswith 10 μM. CDDP or 4 gray γ-irradiation (IR) (I). Representativeimmunoblots of 3 independent experiments are shown. (J) Effects ofpharmacological inhibition of P2Y2 on cellular cannibalism induced byreplicative stress. Replicative stressed human primary fibroblasts werestained, mixed and cultured during 24 hours in presence or in absence ofthe 20 μM of P2Y2 inhibitor Kaempferol. The frequencies of cell-in-cellstructures were determined for at least 300 cells in 3 independentexperiments (mean+/−s.e.m, p<0.001). (K) Effects of P2Y2 depletion oncellular cannibalism observed after p53 or Δ133p53 depletions or p53deletion. As previously described, HCT116 cells that are depleted forP2Y2 and/or inactivated for p53 or Δ133p53 were stained, mixed andcultured during 24 hours in presence or in absence of 20 μM of Y27632 or100 μM of zVAD. The frequencies of cell-in-cell structures weredetermined for at least 300 cells in 3 independent experiments(mean+/−s.e.m, *p<0.001).

FIG. 12. Identification of entescence as a non-cell autonomoussenescence. (A) Effects of P2Y2 inhibition on SA-β Gal activity of humanprimary keratinocytes. After the fifth passages, human primarykeratinocytes were cultured during 24 hours in presence or in absence of20 μM of P2Y2 inhibitor Kaempferol. The number of SA-β Gal positivesingle or cannibal cells per total number of cells examined (at least300 per well) was recorded and frequencies of SA-β Gal positive weredetermined. Error bars represent means±SEM (n=3; *p <0.01). (B) Effectsof P2Y2 inhibition on SA-β Gal activity detected after g-irradiation ormithoxantron treatment. HCT116 cells were treated with 10 μM CDDP or 4gray γ-irradiation (IR) and incubated during 24 hours in presence or inabsence of 20 μM of P2Y2 inhibitor Kaempferol. Then as described abovefrequencies of SA-β Gal positive were determined (means±SEM; n=3; *p<0.01). (C) Effects of P2Y2 depletion on SAT Gal activity observed aftercoculture of p53^(−/−) HCT116 cells. As previously described, the numberof SA-β Gal positive single or cannibal cells per total number of cellsexamined (at least 300 per well) was recorded (means±SEM; n=3; *p<0.01). (E) Detection of DNA damage response (DDR) foci and p21^(WAF1)expression in single and cannibal cells after p53 or Δ133p53inactivation. DNA damage response (DDR) foci were identified by γ-H2AX(green), β-catenin (red) and Hoechst 33342 (blue) staining ofinternalizing HCT116 cells. P21WAF1 expression was also analyzed usingantibody against p21^(WAF1) (green). Images are representative of atleast three independent experiments (scale bar: 5 μm). (F-H) Effects ofP2Y2 depletion on DNA damage foci formation, p21^(WAF1) expression andSA-β Gal activity detected after Δ133p53. After P2Y2 depletion, thepercentage of Δ133p53 depleted cells showing positivity for γ-I-H2AX⁺ orp21^(WAF1) expression were determined by confocal microscopy (G,H) andanalyzed for p Gal activity (Means±SEM; n=3; *P <0.01).

FIG. 13. Detection of Entescence in vivo. (A) Representative image ofprimary breast adenocarcinoma stained for β-catenin (green) and nuclei(blue) is shown (a). Phase contrast is also shown (b). Arrow markscannibal cell.π and x respectively internalized nucleus and cannibalcell nucleus. (B) Representative image of primary breast adenocarcinomastained for β-catenin (green), Lamp2 (red) and nuclei (blue) is shown in(a). Arrows indicate cannibal cells. Magnification of cannibal cell isshown in (b). (C) Quantification of cell-in-cell figures in normalprimary breast (n-10) and in primary breast adenocarcinoma (n=30)biopsies (p<0.01). (D) Quantification of cell-in-cell figures in primarybreast adenocarcinoma diagnosed for histological grade I (n=10), gradeII (n=10) and grade III (n=10). (E) Representative images of primarybreast adenocarcinoma obtained from patients treated with neo-adjuvanttreatments stained by hematoxylin and eosin (HE). Four arrows markcell-in-cell figures in (a). Magnifications of two representative imagesare shown in (b) and (c), (F) Representative image of primary breastadenocarcinoma biopsies obtained from patients treated with neo-adjuvanttreatments stained for β-catenin (green), p2^(WAF1) (red) and nuclei(blue) is shown. (G) Quantification of p21^(WAF1) positive (p21^(WAF1+))single cells or cell-in-cell figures detected on neo-adjuvant treated(n=15) or untreated primary breast adenocarcinoma biopsies (n=30)(p<0.001).

FIG. 14. Levels of cellular cannibalism and entescence could help forthe prediction of treatment efficiency in breast cancer, (A)Quantification of cell-in-cell figures in primary breast adenocarcinomabiopsies obtained from patients that are good (n=12) or bad (n=14)responders to neo-adjuvant treatment (p<0.001). (B) Kaplan-Meier diseasefree survival of neo-adjuvant treated patients. (C) Kaplan-Meier overallsurvival of neo-adjuvant treated patients. (D) Quantification ofcell-in-cell figures in primary triple negative breast adenocarcinomabiopsies obtained from patients that are good or bad responders toneo-adjuvant treatment (p<0.001). (E) Kaplan-Meier disease free survivalof neo-adjuvant treated patients. (F) Kaplan-Meier overall survival ofneo-adjuvant treated patients.

FIG. 15. Depletion of Δ133TP53 isoform induces cellular cannibalism inp53 mutated cells. (A) Detection of cell-in-cell structures induced bydepletion of Δ133TP53 in HT29 (p53^(R273H)) cells, as visualized byconfocal microscopy. Cell-in-cell structures were identified byb-catenin (white), CMFDA (green) and CMTMR (red) staining ofinternalizing HCT116 cells. Images are representative of at least fourindependent experiments (scale bar in a, 5 μm and scale bar in b, 1 μm).(B) Frequencies of cell-in-cell structures induced by Δ133TP53knockdown, in HT29 cells in presence or in absence of 20 μm of ROCKinhibitor (Y27632) or 100 μm of pan-caspase inhibitor (zVAD). Thefrequencies of cell-in-cell structures were determined for at least 300cells in 3 independent experiments (mean+/−s.e.m, p<0.001). Images arerepresentative of at least four independent experiments (scale bar, 5μm). (C) Effect of apyrase on cellular cannibalism. Cocultures ofΔ133TP53 depleted HT29 cells in presence of different concentrations ofapyrase were performed. Then, detection of cell-in-cell structures wasperformed by confocal microscopy (mean +/−s.e.m, n=3, p<0.001). (D)Effects of P2Y2 depletion on cellular cannibalism observed afterΔ133TP53 depletion. As previously described, HT29 cells that aredepleted for P2Y2 and/or inactivated for Δ133TP53 were stained, mixedand cultured during 24 hours. The frequencies of cell-in-cell structureswere determined for at least 300 cells in 3 independent experiments mean+/−s.e.m, p<0.001). (E) Effects of cellular cannibalism on cellproliferation. After cell sorting, cell proliferation of Δ133TP53knocked down single or cannibal HT29 cells were performed during 6 days.(F-I) Effects Δ133TP53 depletion on cellular cannibalism and onsenescence induction of HT29 cells. Cannibal cells mediated Δ133TP53knockdown were examined in EdU incorporation assay (F), in p21expression assay (F) and in SA-β-Gal assay (G). (F) Determination of EdUincorporation on cannibal cells induced by Δ133TP53 knockdown.Cell-in-cell structures were identified by β-catenin (red) and Hoechst33342 (blue) staining of internalizing HT29 cells, EdU positive cellsare green. Images are representative of at least three independentexperiments (scale bar: 5 μm). The detection of p21^(WAF1) expression insingle and cannibal HT29 cells after Δ133TP53 inactivation was alsoshown in (F). P21^(WAF1) expression (white), β-catenin (red) and Hoechst33342 (blue) staining of internalizing HCT116 cells is shown. Images arerepresentative of at least three independent experiments (scale bar: .5μm). Arrow show cannibal cells. (G) Representative picture of SA-β Galstaining of single and cannibal cells obtained after Δ133TP53inactivation is shown. Arrow indicates cannibal cell (H) Determinationof EdU incorporation in single and cannibal cells after Δ133TP53inactivation. The number of EdU positive single or cannibal HT29 cellsper total number of cells examined (at least 300 per well) was recorded(n=3). (F) Quantification of p21^(WAF1) positive cells and SA-β Galpositive cells obtained after Δ133TP53 inactivation in cannibal cellsafter TP53 Δ133TP53 inactivation are shown. The percentage of p21^(WAF1)cells or SA-β Gal cells were determined by confocal microscopy or bylight microscopy. Error bars represent means±SEM (n=3; P <0.001).

DETAILED DESCRIPTION OF THE INVENTION

Cancer cells are characterized by several acquired capabilities thatallow them to sustain proliferative signaling, to evade grog hsuppressors, to resist to cell death, to enable replicative immortality,to reprogram energy metabolism, to induce angiogenesis, to escape immunesystem and to activate invasion (and metastasis) {Hanahan, Cell 2000;Hanahan, Cell 2011}. Signaling interactions between cancer cells and thetumor microenvironment cells (such as fibroblasts or immune cells) alsocontribute to cancer pathogenesis {Hanahan, Cell 2011}. Inactivation oftumor suppressor genes (such as p53) that is frequently detected inhuman tumors {Hanahan, Cell 2000; Hanahan, Cell 2011} contributes to theacquisition of theses cancer cell capabilities, but also impacts tumorpathogenesis by modulating signaling networks in the tumormicroenvironment {Hanahan, Cell 2011}.

The tumor suppressor gene p53 (that is mutated in approximately half ofhuman tumors) promotes a variety of cellular responses depending on thetype of tissue, the nature of the stress signal and the cellularmicroenvironment {Vousden, Nature reviews Molecular cell biology 2009}.P53 promotes cell survival activity through the activation of survivalsignaling pathways {Janieke, Cell Death Differ 2008}, the protectionagainst DNA damage {Bensaad, Cell 2006}, the modulation of energymetabolism {Tolstonog, PNAS 2010; Gottlieb, Cold Spring Harb PerspectBiol 2010} and antioxidant activities {Sablina, The Journal ofbiological chemistry 2005}. In addition, the signaling pathwaysoperating upstream or downstream p53 can be interrupted in numeroustumors, suggesting that the pathways organized around p53 are criticalfor oncogenesis and tumor progression {Vogelstein, Nature 2000}. Inresponse to a wide range of cellular stresses (including genotoxicdamages, deregulated oncogenes, loss of cell contacts and hypoxia), p53provides the elimination of cancer cells by stopping their developments(through anti-angiogenic activities {Tasdemir, J Mol Med 2007} orinhibition of their migration and invasion functions {Gadea, EMBO J.2002; Gadea, J Cell Biol. 2007; Cartier-Michaud, PLoS One 2012}), byinducing cell death {Yonish-Rouach, Nature 1991} or by promotingsenescence {Vousden, Cell 2007; Vousden, Nature reviews Cancer 2002}).Recent counterintuitive works reveal that p53 may also exert itsactivity through non-cell autonomous function by modifying senescentassociated secreted profit (SAP) secretion {Coppe, PLoS Biol 2008} andby repressing in some circumstances cellular senescence {Demidenko, PNAS2010}.

The N-terminal isoforms that lacked the transactivating domain (Δ40TP53and Δ133TP53) act as dominant-negative regulators of p53 activity, andΔ133TP53 silencing has been associated with replication-inducedsenescence in normal human fibroblasts through enhanced transcriptionalregulation of p53-target genes, such as p21^(WAF1) and mir-34a (Fujitaet al., Nat Cell Biol 2009).

Despite the diversity of functions of p53, the role of p53 in cellularcannibalism has never been studied.

In this context, the present inventors show here for the first time thatthe tumor suppressor TP53 and its Δ133TP53 isoform act as repressors ofcellular cannibalism. Indeed, loss of TP53 or Δ133TP53 expressionincreases extracellular ATP release and the consequent activation ofpurinergic P2Y2 receptors which signals for engulfment. They furtherdemonstrate that cannibal cells activate a senescence program throughp2^(WAF1) induction, unrevealing a new modality of induction of cellularsenescence that can occur in the absence of TP53 or Δ133TP53. Senescenceinduced by oncogenic Ras and by replicative or oxidative stresses alsoresults in cellular cannibalism, unrevealing that cannibalism is acommon feature of senescent cells. Accordingly, cannibal cells found inhuman breast carcinomas exhibited signs of p21^(WAF1) activation.Altogether, these results provide evidence that cellular cannibalism andsenescence are tightly linked in human cancer. Moreover, the presentinventors reveal that loss of p53 triggers an unsuspected senescentprocess that requires cell-in-cell internalization and entoticmechanisms to occur. This non-cell autonomous senescent process wascalled “entescence”. It is observed during senescence-induced stresses(such oncogenic stimuli, replicative stresses, chemo- or radiotherapies)and in human tumors. In addition, they also demonstrate that senescenceinduced in cell autonomous manner also results in cellular cannibalismrevealing that cellular cannibalism is a common feature of senescentculls. These results underscore the interplay between cellularcannibalism and senescence.

Definitions

For performing the different steps of the method of the presentinvention, there may be employed conventional molecular biology,microbiology and recombinant DNA techniques within the skill of the art.Such techniques are explained fully in the literature. See, for example,Sambrook, Fitsch & Maniatis, Molecular Cloning: A Laboratory Manual,Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (referred to herein as “Sambrook et al., 1989”); DNACloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985);Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization(B. D. Flames & S. J. Higgins, eds. 1984); Animal CellCulture (R. I. Freshney, ed. 1986); Immobilized Cells and Enzymes (IRL,Press, 1986); B. E. Perbal, A Practical Guide to Molecular Cloning(1984); F. M. Ausubel et al. (eds.), Current Protocols in MolecularBiology, John Wiley & Sons, Inc. (1994).

p53 (hereafter also referred to as TP53) is a protein of apparentmolecular weight 53 kDa that functions as a transcription factor that,among other functions, regulates the cell cycle and functions as a tumorsuppressor as mentioned above. Other isoforms or variants of p53 havebeen identified (see Bourdon, Brit. J. Cancer, 2007). For example, twoother members of p53 family, p63 and p73, which are encoded by distinctgenes, have been identified (Kaghad et al, Cell 1997; and Yang et al.Mol. Cell 1998). Human p53 isoforms may also arise due to alternativepromoter usage and alternative Alternative promoter usage, for example,can give rise to the expression of an N-terminally truncated p53 proteininitiated at codon 133 (Δ133p53 or Δ133TP53). Adding to the complexityof p53 isoforms is the alternative splicing of intron 9 of the p53 geneto provide the isoforms p53β and p53γ. Combined with alternativepromoter usage, this gives rise to the p53 isoforms: p53, p53β(p53beta), p53γ (p53gamma), Δ133p53 (delta133p53), Δ133p5313(delta133p53beta), and Δ133p53γ (delta33p53gamma). The use of analternative promoter in intron 2 gives rise to the additional isoforms,Δ40p53 (delta40p53), Δ40p53β (delta40p53beta), and Δ40p53γ(delta40p53gamma). While the presence of these multiple p53 isoforms hasbeen established, the biological function of these isoforms remainsobscure. The present invention is based in part on an elucidation of therole for p53 and three of these isoforms, Δ133p53, p53β and p53γ, in thefunctions of cell senescence and cell cannibalism.

As used herein, the term “p53” refers generally to a protein of apparentmolecular weight of 53 kDa on SDS PAGE that functions as the tumorsuppressor described above. The protein and nucleic sequences of the p53protein from a variety of organisms from humans to Drosophila are knownand are available in public databases, such as in accession numbers,NM_000546 (SEQ ID NO:1), NP_000537 (SEQ ID NO:11, NM₀₁₁₆₄₀ (SEQ ID NO:2)and NP_035770 (SEQ ID NO:12, for the human and mouse sequencesrespectively. It is also referred to as “p53α” or “p53alpha”. Itcontains an entire transactivation domain (including TAD1, and TAD2),the PXXP domain, a DNA binding domain, the NLS and an entireoligomerisation domain in C-terminal.

The term “Δ133p53” or “delta133p53” or “Δ133TP53” or “delta133TP53”refers generally to the isoform of p53 that arises from initiation oftranscription of the p53 gene from codon 133, which results in anN-terminally truncated p53 protein. This isoform comprises the followingp53 protein domains: the majority of the DNA binding domain, the NLS,and the C-terminal sequence DQTSFQKENC see Bourdon, Brit. J. Cancer,2007). It has for example SEQ ID NO:14. It is encoded for example by SEQID NO: 3.

The term “p53β” or “p53beta” refers generally to the isoform of p53 thatarises from alternative splicing of intron 9 to provide a p53 isoformcomprising the following p53 protein domains: TAD1, TAD2, prD, the DNAbinding domain, the NLS, and the C-terminal sequence DQTSFQKENC (seeBourdon, Brit. J, Cancer, 2007). It has for example the sequence SEQ IDNO:15. It is encoded for example by SEQ ID NO: 4.

The term “p53γ” or “p53gamma” refers generally to the isoform of p53that arises from alternative splicing of intron 9 to provide a p53isoform comprising the following p53 protein domains: TAD1, TAD2, prD,the DNA binding domain, the NLS, and the C-terminal sequenceMLLDLRWCYFLINSS. It has for example the sequence SEQ ID NO:16. It isencoded for example by SEQ ID NO: 5.

The term “Δ40p53” or “Δ40TP53” or “delta40p53” or “delta40TP53” refersgenerally to the isoform of p53 that arises from whole splicing ofintron 9, no splicing of intron 2 and normal splicing of exons 1,3 and11 to provide a p53 isoform comprising the following p53 proteindomains: TAD2, PXXP, the DNA binding domain, the NLS, and the entireoligomerisation domain in C-terminal. It has for example the sequenceSEQ ID NO:13. It is encoded for example by SEQ ID NO: 6.

The term “promoting” as used, for example in the context of “promotingcannibalism,” refers generally to conditions or agents which increase,induce, open, activate, facilitate, enhance activation, sensitize,agonize, or up regulate cell cannibalism.

“Inhibitors,” “activators,” and “modulators” of cellular cannibalism areused to refer to activating, inhibitory, or modulating moleculesidentified using the in vitro of the invention. Inhibitors are compoundsthat, decrease, prevent, or down regulate the expression of p53isoforms. “Activators” are compounds that increase, facilitate, or upregulate activity of p53 isoforms. Inhibitors, activators, or modulatorsinclude naturally occurring and synthetic ligands, antagonists,agonists, antibodies, peptides, cyclic peptides, nucleic acids,antisense molecules, ribozymes, RNAi molecules, small organic moleculesand the like. Such assays the inhibitors and activators include, e.g.,expressing p53 isoforms in vitro, in cells, or cell extracts, applyingputative modulator compounds, and then determining the functionaleffects on p53 expression, as described below. The term “test compound”or “drug candidate” or “modulator” or grammatical equivalents as usedherein describes any molecule, either naturally occurring or synthetic,e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acidsin length, preferably from about 10 to 20 or 12 to 18 amino acids inlength, preferably 12, 15, or 18 amino acids in length), small organicmolecule, polysaccharide, peptide, circular peptide, lipid, fatty acid,siRNA, polynucleotide, oligonucleotide, etc., to be tested for thecapacity to directly or indirectly modulate p53 isoforms. The testcompound can be in the form of a library of test compounds, such as acombinatorial or randomized library that provides a sufficient range ofdiversity. Test compounds are optionally linked to a fusion partner,e.g., targeting compounds, rescue compounds, dimerization compounds,stabilizing compounds, addressable compounds, and other functionalmoieties. Conventionally, new chemical entities with useful propertiesare generated by identifying a test compound (called a “lead compound”)with some desirable property or activity, e.g., inhibiting activity,creating variants of the lead compound, and evaluating the property andactivity of those variant compounds. Often, high throughput screening(HTS) methods are employed for such an analysis.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 daltons and less than about 2500 daltons, preferably lessthan about 2000 daltons, preferably between about 100 to about 1000daltons, more preferably between about 200 to about 500 daltons.

Most of the methods of the invention are performed in vitro. Asdisclosed herein, the terms “in vitro” and “ex vivo” are equivalent andrefer to studies or experiments that are conducted using biologicalcomponents (e.g. cells or population of cells) that have been isolatedfrom their usual host organisms (e.g. animals or humans). Such isolatedcells can be further purified, cultured or directly analyzed to assessthe presence of the mutant proteins. These experiments can be forexample reduced to practice in laboratory materials such as tubes,flasks, webs, eppendorfs, etc. In contrast, the term “in vivo” refers tostudies that are conducted on whole living organisms.

The screening methods of the invention are preferably performed on cellsamples expressing p53 isoforms. In the context of the invention, thesecell samples contain for example cell lines that are known to expressone or the other isoforms. In a preferred embodiment, these cell linesare primary human diploid WI38 fibroblasts or cancer cell lines such ascolorectal HCT116 carcinoma or pancreatic PANC813 cells.

The prognosis methods of the invention are preferably performed on abiological sample obtained from a patient in need thereof. As used inthe context of these methods, the term “biological sample”advantageously refers to a serum sample, a plasma sample, a bloodsample, a lymph sample, or to a tumor tissue sample obtained by biopsy.Preferably, the said biological sample is a blood sample or a tumortissue sample obtained from a tumor biopsy. Said tissue sample is forexample a tumor sample obtained from a primary breast adenocarcinoma, aprimary cervical adenocarcinoma, a primary pancreatic adenocarcinoma, aprimary melanoma, primary spitz tumors, a primary stomach or livercarcinoma.

The prognosis method of the invention can include the steps consistingof obtaining a biological sample from said subject and extracting thenucleic acid from said biological sample. The DNA can be extracted usingany known method in the state of the art. The RNA can also be isolated,for example from tissues obtained during a biopsy, using standardmethods well known to those skilled in the art, such as extraction byguanidium-thiophenate-phenol-chloro form.

The expression level of a protein in a biological sample is determinedby:

-   a method including a PCR, qPCR, a RT-PCR method, in situ    hybridization, a Northern method or microarrays when the expression    level of RNA transcripts encoding a defined protein is to be    determined; or-   Enzyme linked immunosorbent assays (ELISAs), Western blots,    immunoprecipitations, immunofluorescence method, electron method or    Enzyme-linked staining method for microscopic detection    immunohistochemistry assay), or flow cytometry, when the expression    level of the protein itself is to be determined.

Such methods are well known from the skilled person (see e.g., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

In a preferred embodiment, quantitative RT-PCR (qPCR) analysis can beused to measure the mRNA expression levels in a biological sample. Geneexpression analysis by real-time quantitative PCR (RT-qPCR) is wellknown from the skilled person. For example, p53 mRNA expression analysisby RT-qPCR can be assessed after standard tissue sample RNA extraction(for example, the samples can be lysed in a tris-chloride, EDTA, sodiumdodecyl sulphate and proteinase K containing buffer; RNA can be thenextracted with phenol-chloroform-isoamyl alcohol followed byprecipitation with isopropanol in the presence of glycogen and sodiumacetate; RNA can be resuspended in diethyl pyrocarbonate water (AmbionInc., Austin, Tex.) and treated with DNAse 1 (Ambion Inc., Austin, Tex.)to avoid DNA contamination; complementary DNA can be synthesized usingfor example Maloney Murine Leukemia Virus retrotranscriptase enzyme;template cDNA can be added to Taqman Universal Master Mix (AB, AppliedBiosystems, Foster City, Calif.) with specific primers and probe foreach p53 isoforms.

The primer and probe sets can be designed using Primer Express 2.0Software (AB) and the reference sequences (which can be obtained on theweb site http://www.ncbi.nlm.nih.gov/entrez/query. fcg,i?db=gene).Nucleic acid probes and primers for hybridizing specifically the mRNA orcDNA of p53 isoforms are well-known in the art. They are typically of atleast 10, 15 or 20 nucleotides in length that is sufficient tospecifically hybridize under stringent conditions to the mRNA or cDNA ofp53 isoforms, or complementary sequence thereof (preferred areoligonucleotide primers or probe having at least 90%, 95%, 99% and 101)% identity with the mRNA sequence fragment of the p53 isoforms or thecomplementary sequence thereof).

Protein expression levels can be for example detected using antibodiesspecifically directed against different specific regions or epitope ofthe p53 protein and isoforms thereof. The term “antibody” as used hereinrefers to immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (or immunorcacts with) the p53protein or isoforms thereof. The term “antibody” comprises monoclonal orpolyclonal antibodies but also chimeric or humanized antibodies. Theseantibodies preferably differently bind the different isoforms of p53.These antibodies are for example the commercial mouse monoclonal DO-1and DO-7 (recognizing specifically the p53P and p53γ isoforms), thecommercial mouse monoclonal 1801 (recognizing all p53 isoforms exceptΔ133p53, the commercial mouse monoclonal antibodies BP53.10, 421, andICA-9 (that are specific for the α isoforms of p53 (p53, Δ40p53, andΔ133p53), because their epitopes are localized in the basic region (BR)of the p53 protein), and the rabbit polyclonal antibodies KJC8(recognizing specifically p53β) and MAP4.9 (recognizing specificallyΔ133p⁵ see Khoury M. P. and Bourdon J. C. Cold Spring Perspect Biol.2010).

Preferably, the antibodies and probes used in the methods of theinvention are labeled so as to be detected.

The term “labeled” with regard to a probe or an antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin.

In the screening methods of the invention, cells expressing various p53isoforms are treated with potential activators, inhibitors, ormodulators so as to examine the extent of p53 isoform expression levelmodulation. In this case, “control levels” correspond to p53 isoformexpression levels and/or ATP release and/or P2Y2R expression or activitylevels measured in control cells, said control cells preferably beingthe same cells that have not been treated with said activators,inhibitors, or modulators. Therefore, control levels correspond to p53isoform and/or ATP and /or P2Y2R expression levels measured in nontreated cells.

In a particular embodiment, control samples (untreated) can be assigneda relative protein expression value of 100%. In this case, decrease (orrepression) of the expression (or expression level) of a target proteinwould be achieved when the expression level of said protein is of about80%, preferably 50%, more preferably 25-0% relative to the controllevel. Conversely, increase (or promotion) of the expression (orexpression level) of a target protein would be achieved when theexpression level of said protein is at least of about 110%, morepreferably at least of about 150%, and is more preferably of 200-500%(i.e., two to five fold superior to the control), more preferably of1000-3000% relative to the control level.

Also, as used in the present application, a “low” expression of a targetprotein is achieved when the expression level of said protein is lessthan about 80%, preferably less than about 50%, more preferably lessthan about 25% relative to the control level. Conversely, a “high”expression of a target protein is achieved when the expression level ofsaid protein is at least of about 110%, more preferably at least ofabout 150%, and is more preferably of 200-500% relative to the controllevel, i.e., from two to five folds superior to the control level.

In the prognosis methods of the invention, the p53 isoform or P2Y2Rexpression levels measured in the tumor cells of the patients arecompared with “control level(s)”. These control levels are preferablymeasured in normal cells or in non-treated tumor cells of the samepatient, more preferably in normal cells of at least one anothersubject, preferably of an healthy subject.

Adenosine-5′-triphosphate (ATP) is a nucleoside triphosphate used in allliving cells for intracellular energy transfer. It is one of the endproducts of photophosphorylation, cellular respiration, and fermentationand used by enzymes and structural proteins in many cellular processes,including biosynthetic reactions, motility, and cell division. Onemolecule of ATP contains three phosphate groups. The structure of thismolecule consists of a purine base (adenine) attached to the carbon atomof a pentose sugar (ribose). Three phosphate groups are attached at the5′ carbon atom of the pentose sugar. It is produced by a wide variety ofenzymes, including ATP synthase, from adenosine diphosphate (ADP) oradenosine monophosphate (AMP) and various phosphate group donors.

ATP has the formula I

ATP is typically quantified by measuring the light produced through itsreaction with the naturally occurring firefly enzyme luciferase using aluminormeter. The amount of light produced is directly proportional tothe amount of ATP present in the sample.

Extracellular ATP can be measured easily by a number of commercial kits(e.g., the Enliten ATP assay system of Promega). High-Performance liquidchromatography (HPLC) may also be used to measure extracellular levelsof ATP.

The P2Y2 purinergic receptor, also known as “P2Y purinoceptor 2” or“P2Y2R” is a protein that in humans is encoded by the P2RY2 gene locatedon the chromosome 11 (Parr C E. et al, 1994, PNAS 1991), This receptorbelongs to the family of G-protein coupled receptors. It favors theproduction of both the adenosine and uridine nucleotides. It mayparticipate in control of the cell cycle of several cancer cells. Inhuman, three transcript variants encoding the same protein have beenidentified for this gene. These transcripts have the sequence SEQ IDNO:7 (variant 1) SEQ ID NO:8 (variant 2) and SEQ ID NO:9 (variant 3).The human P2Y2 purinergic receptor has the polypeptide sequence SEQ IDNO:10.

siRNAs are described for example in WO 02/44 321 (MIT/MAX PLANCKINSTITUTE). This application describes a double strand RNA (oroligonucleotides of same type, chemically synthesized) of which eachstrand has a length of 19 to 25 nucleotides and is capable ofspecifically inhibiting the post-transcriptional expression of a targetgene via an RNA interference process in order to determine the functionof a gene and to modulate this function in a cell or body. Also, WO00/44895 (BIOPHARMA) concerns a method fir inhibiting the expression ofa given target gene in a eukaryote cell in vitro, in which a dsRNAformed of two separate single strand RNAs is inserted into the cell, onestrand of the dsRNA having a region complementary to the target gene,characterized in that the complementary region has at least 25successive pairs of nucleotides. Persons skilled in the art may refer tothe teaching contained in these documents to prepare the siRNAs of theinvention.

MicroRNAs (hereafter referred to as miRNAs) are small non-coding RNAmolecule (ca. 22 nucleotides) found in plants and animals, whichfunctions in transcriptional and post-transcriptional regulation of geneexpression, miRNAs function via base-pairing with complementarysequences within mRNA molecules, usually resulting in gene silencing viatranslational repression or target degradation, miRNAs have beenproposed in therapeutic strategies for treating cancer and acuteischemic diseases (Li C. et al, AAPSJ, 2009; .Fasanaro et al, Pharmacol.Ther. 2010).

By “pharmaceutically acceptable vehicle”, it is herein designated anyand all solvents, buffers, salt solutions, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. The type ofcarrier can be selected based upon the intended route of administration.In various embodiments, the carrier is suitable for intravenous,intraperitoneal, subcutaneous, intramuscular, topical, transdermal ororal administration. Pharmaceutically acceptable carriers includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of media and agents for pharmaceutically activesubstances is well known in the art.

An “effective amount” herein refers to an amount that is effective, atdosages and for periods of time necessary, to achieve the desiredresult, i.e., to treat effectively the patient. An effective amount asmeant herein should also not have any toxic or detrimental severeeffects.

Screening Methods for Identifying Modulators of Cellular Cannibalism andSenescence

In a first aspect, the present invention provides methods foridentifying compounds that modulate cell cannibalism via its effect onp53 or Δ133p53, In general, the method includes these steps: (a)contacting a candidate compound with a sample that comprises p53 orΔ133p53, and (b) determining the functional effect of the candidatecompound on the expression level of p53 or Δ133p53, based on which onemay determine whether the said compound is an activator or an inhibitorof cellular cannibalism.

In one embodiment, the present invention relates to a method ofidentifying a compound that modulates cellular cannibalism, comprisingthe steps of:

-   (a) adding a compound to a cell culture in vitro,-   (b) measuring in said culture the expression level of a protein    selected from the group consisting of p53 and N-terminal isoforms of    p53 that lack the N-terminal transactivating domain, such as Δ40TP53    and Δ133TP53;-   wherein a compound that represses cellular cannibalism is identified    by a normal expression or an increase in the expression levels of    p53 or said isoforms, as compared to control levels.

Preferably, the said compound increases cellular cannibalism if theexpression level of p53 or said isoforms decreases, as compared tocontrol levels.

By “normal expression”, it is herein meant that the expression of p53 orof its N-terminal isoforms lacking the N-terminal transactivating domainis almost identical 5%) to the control levels.

More preferably, in this embodiment, said cell culture contains orconsists of primary human diploid WI38 fibroblasts, or cancer cell linessuch as colorectal HCT116 carcinoma or pancreatic ductal PANC813adenocarcinoma.

Even more preferably, in this method, p53 is the polypeptide of SEQ IDNO11, Δ40TP53 is the polypeptide of SEQ ID NO:13, and Δ133TP53 is thepolypeptide of SEQ ID NO:14.

In another embodiment, the present invention relates to a method ofidentifying a compound that modulates cellular cannibalism, comprisingthe steps of:

-   (a) adding a compound to a cell culture in vitro,-   (b) measuring in said culture the expression level of a protein    selected from the group consisting of p53β and p53γ,-   wherein a compound that represses cellular cannibalism is identified    by a decrease in the expression levels of p53β and/or p53γ, as    compared to control levels.

Preferably, the said compound increases cellular cannibalism if theexpression levels of p53β and p53γ are increased as compared to controllevels.

More preferably, s in this embodiment, said cell culture contains orconsists of primary human diploid WI38 fibroblasts or cancer cell linessuch as colorectal HCT116 carcinoma or pancreatic ductal PANC813adenocarcinoma.

Even more preferably, in this method, p53β is the polypeptide of SEQ IDNO:15 and is the polypeptide of SEQ ID NO:16.

In another embodiment, the present invention relates to a method ofidentifying a compound that modulates cellular cannibalism, comprisingthe steps of:

-   (a) adding a compound to a cell culture in vita-   (b) measuring in said culture the extracellular amount of ATP,-   wherein a compound that represses cellular cannibalism is identified    by a decrease in the extracellular amount of ATP, as compared to    control levels.

Preferably, said compound increases cellular cannibalism if theextracellular amount of ATP, is increased as compared to control levels.

More preferably, in this embodiment said cell culture contains orconsists of primary human diploid WI38 fibroblasts or cancer cell linessuch as colorectal HCT116 carcinoma or pancreatic ductal PANC813adenocarcinoma.

In another embodiment, the present invention relates to a method ofidentifying a compound that modulates cellular cannibalism, comprisingthe steps of:

-   (a) adding a compound to a cell culture in vitro,-   (b) measuring in said culture the expression level or the activity    of the purinergic P2Y2 receptor,-   wherein a compound that represses cellular cannibalism is identified    by a decrease in the expression level car in the activity of the    purinergic P2Y2 receptor, as compared to control levels.

Preferably, said compound increases cellular cannibalism if theexpression level or the activity of the purinergic P2Y2 receptorincreases, as compared to control levels.

In this method, the expression level of purinergic P2Y2 receptor may bemeasured as defined above, that is, by measuring the mRNA level or theprotein level of the said receptor in the cells.

Alternatively, this method may involve the measurement of the activityof the purinergic P2Y2 receptor. By “activity”, it is herein mean the“biological activity” of the said receptor. P2Y2 receptor biologicalactivity is triggered by the binding of extracellular nucleotides (suchas ATP and UTP) and is coupled to intracellular signaling pathwaysthrough heterotrimeric G proteins activation and formation of apolyprotein complex that activates kinases (such as the proline-richtyrosine kinase 2 (Pyk2)) involved in numerous cellular functions likeplasma membrane permeabilization, Ca⁺ influx and cytoskeletonreorganization. It can be detected for example by determining thephosphorylation of Pyk2 on tyrosine 402, one cellular consequence ofP2Y2 biological activity (Séror et al., The Journal of e medicine 2011).

More preferably, in this embodiment, said cell culture contains orconsists of primary human diploid WI38 fibroblasts or cancer cell linessuch as colorectal HCT116 carcinoma or pancreatic ductal PANC813adenocarcinoma.

Even more preferably, in this method, the purinergic P2Y2 receptor isthe polypeptide of SEQ ID NO:10.

Of course, a combination of the above-mentioned steps can be used toidentify compounds that modulate cellular cannibalism.

Consequently, the present invention also relates to a screening methodcomprising the step of a cell culture:

-   -   the expression level of protein selected from the group        consisting of p53 and N-terminal isoforms of p53 that lack the        N-terminal transactivating domain, such as Δ40TP53 and        ΔA133TP53, and    -   the expression level or the activity of the purinergic P2Y2        receptor,

-   wherein the tested compound represses cellular cannibalism if the    expression levels of p53 or said isoforms increase, as compared to    control levels, and if the expression level or the activity of the    purinergic P2Y2 receptor decreases, as compared to control levels.

In a preferred embodiment, the tested compound increases cellularcannibalism if the expression levels of p53 or said isoforms decrease,as compared to control levels, and if the expression level of thepurinergic P2Y2 receptor or the activity increases, as compared tocontrol levels.

More preferably, in this embodiment, said cell culture contains orconsists of primary human diploid WI38 fibroblasts or cancer cell linessuch as colorectal HCT116 carcinoma or pancreatic ductal PANC813adenocarcinoma.

The present invention also relates to a method of identifying a compoundthat modulates cellular cannibalism, comprising the step of measuringcell culture:

-   -   the expression level of a protein selected from the group        consisting of p53 and N-terminal isoforms of p53 that lack the        N-terminal transactivating domain, such as Δ40TP53 and Δ133TP53,        and the extracellular amount of ATP,

-   wherein a compound that represses cellular cannibalism is identified    by an increase in the expression levels of p53 or said isoforms, as    compared to control levels, and a decrease in the extracellular    amount of ATP, as compared to control levels.

In a preferred embodiment, the tested compound increases cellularcannibalism if the expression levels of p53 or said isoforms decrease,as compared to control levels, and if the extracellular amount of ATPincreases, as compared to control levels.

More preferably, in this embodiment, said cell culture contains orconsists of primary human diploid WI38 fibroblasts or cancer cell linessuch as colorectal HCT116 carcinoma or pancreatic ductal PANC813adenocarcinoma.

The present invention also relates to a method of identifying a compoundthat modulates cellular cannibalism, comprising the step of measuring ina cell culture:

-   -   the expression level of a protein selected from the group        consisting of p53 and N-terminal isoforms of p53 that lack the        N-terminal transactivating domain, such as Δ40TP53 and Δ133TP53,        the expression level or the activity of the purinergic P2Y2        receptor and the extracellular amount of ATP,

-   wherein a compound that represses cellular cannibalism is identified    by an increase in the expression levels of p53 or said isoforms, as    compared to control levels, and a decrease in both the extracellular    amount of ATP, and in the expression level or the activity of the    purinergic P2Y2 receptor, as compared to control levels.

In a preferred embodiment, the tested compound increases cellularcannibalism if the expression levels of p53 or said isoforms decrease,as compared to control levels, and if the extracellular amount of ATPincreases as compared to control levels, and if the expression level orthe activity of the purinergic P2Y2 receptor increases, as compared tocontrol levels.

More preferably, in this embodiment, said cell culture contains orconsists of primary Truman diploid WI38 fibroblasts or cancer cell linessuch as colorectal HCT116 carcinoma or pancreatic ductal PANC813adenocarcinoma.

In a preferred embodiment, measuring expression levels of the p53isoforms protein or of the P2Y2 receptor comprises measuring the mRNAlevels of these proteins. In a more preferred embodiment, measuringexpression levels of the p53 isoform protein or of the P2Y2 receptorcomprises using a transcriptional fusion between said p53 isoform orreceptor and a reporter molecule.

In Vitro Methods for Detecting Cannibalism Behavior of a Cell

So far, this activity was commonly assessed by studying the cells underthe microscope (by immunofluorescence or immunohistochemistry) and byvisually detecting cell engulfment events or cell-in-cell systems.

The present inventors have shown in the experimental part of the presentapplication that it is possible to detect early events of a cannibalismbehavior of a cell, by measuring, in said cell, the expression level ofa protein selected from the group consisting of: p53, p53β, p53γ, andN-terminal isoforms of p53 that lack the N-terminal transactivatingdomain, such as Δ40TP53 and Δ133TP53, or by measuring the expressionlevel or the activity of the purinergic P2Y2 receptor, and/or bymeasuring the extracellular ATP secreted by said cells.

Study of these molecular pathways therefore results in a fine analysisof the ea mechanisms resulting in cannibalistic activity, before themorphologic events usually attributed to engulfing activity can beobserved.

Thus, in another aspect, the present invention relates to an in vitromethod for detecting cannibalism behavior of a cell, comprising the stepof measuring in said cell:

-   -   the expression level of a protein selected from the group        consisting of p53 and N-terminal isoforms of p53 that lack the        N-terminal transactivating domain, such as Δ40TP53 and Δ133TP53,        and/or the expression level or the activity of the purinergic        P2Y2 receptor, and/or    -   secreted extracellular ATP, and/or    -   the expression level of a protein selected from the croup        consisting of p53β and p53γ,

-   wherein the said cell has cannibalism activity if the expression    levels of p53 or said Δ40TP53 and Δ133TP53 isoforms is low, and/or    if secretion of extracellular ATP is high, and/or if the expression    level or the activity of the purinergic P2Y2 receptor is high,    and/or if the expression level of p53β or p53γ is high, as compared    to control levels.

Preferably, the said cell has no cannibalism activity if the expressionlevels of p53 or said Δ40TP53 and Δ133TP53 isoforms is high, and/or ifsecretion of extracellular ATP by said cell is low, and/or if theexpression level or the activity of the purinergic P2Y2 receptor is low,and/or if the expression level of p53β or p53γ is low, as compared tocontrol levels.

In a preferred embodiment, the method of the invention further comprisesthe step of measuring at least one senescence marker, for exampleselected from the group consisting of: p21^(WAF1), Ki67, p16, Rb, andSA-β-Gal. It is also possible to detect the senescent morphology of thecells by microscopic analysis, for example the increase of cell andnuclear sizes, the detection of senescent associated heterochromatinfoci or cell autophagy or the markers of growth arrest (for example anincrease in p16), or the secretion of senescent associated secretedproteases (SASP), or markers of DNA damage responses (g-H12AX,ATMS1981P, p53BP1, etc.).

Thus, in a more preferred embodiment, the present invention relates toan in vitro method for detecting simultaneously cannibal and senescentbehavior of a cell, comprising the step of detecting cellularcannibalism markers as mentioned in the present application andsenescent markers disclosed above. More precisely, said method comprisesthe step of measuring, in said cell:

-   -   the expression level of a protein selected from the group        consisting of p53 and N-terminal isoforms of p53 that lack the        N-terminal transactivating domain, such as Δ40TP53 and Δ133TP53,        and/or    -   the expression level or the activity of the purinergic P2Y2        receptor, and/or    -   secreted extracellular APT, and/or    -   the expression level of a protein selected from the group        consisting of p53β and p53γ, and    -   the expression level of at least one senescence marker such as        p21^(WAF1), Ki67, p16, Rb, or SA-β-Gal,

-   wherein the said cell has cannibal activity and undergo senescence    if the expression levels of p53 or said Δ40TP53 and Δ133TP53    isoforms are low, and/or if secretion of extracellular ATP is high,    and/or if the expression level or the activity of the purinergic    P2Y2 receptor is high, and/or if the expression level of p53β or    p53γ is high, and if the expression level of the senescence markers    p21^(WAF1), p16, and/or SA-β-Gal is high or if the expression level    of the senescence markers Ki67 and/or Rb is low compared to control    levels.

In another aspect, the present invention relates to an in vitro methodof determining an increase in the propensity of a cell to be a target ofcellular cannibalism.

The present inventors have indeed observed that any of the followingevents: (a) increased extracellular release of ATP, (b) decreased p53expression, (c) decreased Δ133TP53 expression, (d) increased expressionof p53β or p53γ, (e) increased expression of P2Y2 purigenic receptors,induces an increase in the propensity of a cell to be a target ofcellular cannibalism.

As used herein, the expression “increase in the propensity of a cell tobe a target of cellular cannibalism” means that there is an increase inthe “eat-me” signals emitted by the cells. These signals are for exampleextracellular nucleotides such as ATP or UTP, that are known signals forcell engulfment.

In a preferred embodiment, measuring the expression levels of the saidproteins comprises measuring their mRNA levels, for example by qPCR orby in situ hybridization. Also, secreted ATP is preferably measured byHPLC or luciferase assays. In addition, biological activity of P2Y2R ispreferably measured by determining the phosphorylation of Pyk2 ontyrosine 402 (Séror et al. The Journal of experimental medicine 2011).

In Vitro Methods for Inducing Cellular Cannibalism in a Cell

The present inventors identified for the first time that low levels ofp53, of Δ133TP53 or of Δ40TP53 promotes cellular cannibalism.

In another aspect, the present invention furthermore relates to a methodof inducing cellular cannibalism in a cell in vitro, comprisinginhibiting in said cell the expression of p53 or of an N-terminalisoform of TP53 selected from Δ133TP53 and Δ40TP53.

This inhibition may be obtained by inhibiting the formation of the saidproteins (e.g., by inhibiting the transcription of DNA to mRNA or thetranslation of mRNA to a protein), or by promoting the degradation ofthese proteins by any conventional means.

In particular, said expression inhibition may be mediated by inhibitingthe translation of mRNA to a protein by means of a miRNA or a siRNA.

More specifically, the invention relates to double strand siRNAs ofapproximately 15 to 30 nucleotides, preferably 19 to 25 nucleotides, orpreferably around 19 nucleotides in length that are complementary(strand 1) and identical (strand 2) to nucleotide regions of the p53isoforms implicated in the methods of the invention. These siRNAs of theinvention also comprise a dinucleotide TT or UU at the 3′ end. Numerouscomputer programmes are available for the design of the siRNAs of theinvention.

In one particular embodiment, the siRNAs of the invention are tested andselected for their capability of reducing, even specifically blockingthe expression of one particular p53 isoform, affecting as little aspossible the expression of the other isoforms. For example, theinvention concerns siRNAs allowing a reduction of more than 80%, 90%,95% or 99% of the expression of one p53 isoform, and no reduction or areduction of less than 50%, 25%, 15%, 10% or 5% or even 1% of the otherisoforms of p53.

In a preferred embodiment, said p53 expression inhibition is achieved byusing the siRNA of SEQ ID:17 (siRNA-: 5′GACUCCAGUGGUAAUCUAC 3′) or SEQID NO:18 (siRNA-2: 5′GCAUGAACCGGAGGCCCAU3′).

In another preferred embodiment, said Al 33T1³53 expression inhibitionis achieved by using the siRNA of SEQ IL) NO:19 (siRNA-1:5′UGUUCACUUGUGCCCUGACUUUCAA3′) or SEQ IL) NO:20 (siRNA-2:5′CUUGUGCCCUGACUUUCA3′).

These various siRNAs and miRNAs can be used for inducing simultaneouslycell cannibalism and senescence in cancer cells. Consequently, they canbe used for enhancing the survival of a patient suffering from cancerand, eventually, for treating cancer.

The present invention therefore also relates to the use of these siRNAsand miRNAs for the preparation of a pharmaceutical composition which isintended to treat cancer. Said pharmaceutical composition contains forexample the said siRNAs or miRNAs and a pharmaceutically acceptablevehicle as defined above.

In other words, the present invention relates to a method for treatingcancer, comprising the step of administering, in a patient sufferingfrom cancer, an effective amount of any of the above-mentioned siRNAs ormiRNAs.

These siRNAs can be injected into the cells or tissues by lipofection,transduction or electroporation.

In a related aspect, the present invention relates to a method ofinhibiting cellular cannibalism in a cell in vitro, comprisinginhibiting in said cell the expression of p53β or p53γ, or inhibitingthe expression or the activity of the purinergic P2Y2 receptor, orinhibiting the secretion of extracellular ATP.

In a preferred embodiment, the expression of the isoforms p53β or p53γ,or of the purinergic P2Y2 receptor is inhibited by using a miRNA or asiRNA. P2Y2R expression inhibitors are for example disclosed in Seror C.et al., The Journal of experimental medicine 2011.

Again, these siRNAs can be double strand RNAs of approximately 15 to 30nucleotides, preferably 19 to 25 nucleotides, or preferably around 19nucleotides in length that are complementary (strand 1) and identical(strand 2) to nucleotide regions of the p53 isoforms p53β or p53γ or ofthe purinergic P2Y2 receptor. siRNAs inhibiting the expression of theP2Y2 receptor are for example those of SEQ ID NO:53 or of SEQ ID NO.54.

In one particular embodiment, the siRNAs of the invention are tested andselected for their capability of reducing, even specifically blockingthe expression of p53β or p53γ, affecting as little as possible theexpression of the other p53 isoforms. For example, the inventionconcerns siRNAs allowing a reduction of more than 80%, 90%, 95% or 99%of the expression of p53β or of p53γ, and no reduction or a reduction ofless than 50%, 25%, 15%, 10% or 5% or even 1% of the other isoforms ofp53.

Inhibition of P2Y2R biological activity can be obtained by usingblocking agents such as low molecular weight antagonist (such as a smallorganic P2Y2R inhibitor kaempferol or other purinergic receptorantagonists including but not limited to reactive red 2, suramin,oxidized ATP, 8,8′-(carbonylbis(imino-3,1-phenylenecarbonylimino)bis(1,3,5-naphthalenetrisulfonic acid) (also calledNF023),8,8′-(carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3,5-naphthalenetrisulfonicacid) (also called NF279), Evans blue, trypan blue, reactive blue 2,pyridoxalphosphate-6-azophenyl-29,49-disulfonic acid (PPADS),isoquinoline sulfonamide,1-[N,O-bis(5-isoquinoline-sulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine(also called KN-62), trinitrophenyl-substituted nucleotides (e.g.,TNP-ATP), diinosine pentaphosphate (IP51), cicacron blue 3GA,2′3′-O-(2,4,6-trinitrophenyl)-ATP, substance P (SP), basilen blue, and4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, or combinations,derivatives, or pharmaceutically acceptable salts thereof), blockingantibodies or aptamers or siRNAs.

In another preferred embodiment, the secretion of ATP is inhibited byblocking the membrane channels known to be involved in ATP transport(such as pannexin-1 and connexin-43) or by impairing biologicalmechanisms that are involved in same (such as autophagy or exocytosis)by means of conventional tools.

The present invention also concerns a therapeutic composition comprisinga pharmaceutically acceptable vehicle and at least one of thesecompounds for its use for treating cancer.

Prognosis Methods Based on Detection of Cannibalism and Senescence

Cellular cannibalism and cell senescence were both shown to be involvedin tumorigenesis. However, their precise role with this respect remainscontroversial.

On a one hand, cellular cannibalism was shown to promote tumordevelopment. Tumor cell cannibalism has been therefore linked with poortumor prognosis, what can be due to genomic instability induced in thepolyploid cells (Krajcovic et al., 2011; Krajcovic and Overholtzer,2012.

On another hand, cell senescence was shown to impair tumorigenesis(Canon C E, et EMBO Mol Med. 2012) or to promote same (Krajcovic et al.,Nat Cell Idol 2011; Krajcovic and Overholtzer, Cancer Res 2012), so thatthe measure of senescence markers alone is of poor pronostic value.

The present inventors herein show for the first time that cellularcannibalism and senescence are tightly linked in human cancer and thatthe detection of both processes in cancer cells positively correlateswith good patient's response to treatment. Importantly, their resultsprovide the first evidence that detection of cellular cannibalism andsenescence simultaneously in tumors helps for the diagnosis of diseaseoutcomes and for the prediction of treatment efficiency against cancerdiseases.

In another aspect, the present invention therefore relates to an invitro method of determining the prognosis of cancer in a patient,comprising the step of simultaneously detecting the cannibal andsenescent behavior of the tumor cells of this patient. As a matter offact, if the said tumor cells have simultaneously high cannibal andsenescent activities, then the said tumor cells will be of goodprognosis and the patient survival is likely to be long. In contrastthereto, if the said tumor cells have low (or no) cannibal and senescentactivities, then the said tumor cells will be of bad prognosis and thepatient survival is likely to be short.

In this aspect, the said method preferably comprises the step ofmeasuring the expression levels of p53 or of one of its isoformsΔ133TP53, TP53β, TP53γ, or Δ40TP53, or the expression level or theactivity of the purinergic P2Y2 receptor, and the expression level of asenescence marker such as p21^(WAF1), Ki67, p16, Rb, or SA-β-Gal, or ofany other senescence marker as described above, in tumor cells isolatedfrom the patient.

In fact, if the expression levels of p53 or said Δ40TP53 and Δ133TP53isoforms in said tumor cells are low, or if the expression level or theactivity of the purinergic P2Y2 receptor in said tumor cells is high, orif the expression level of p53β or p53γ in said tumor cells is highcompared to control levels, and, simultaneously, if the expression levelof the senescence markers p21^(WAF1), and/or SA-β-Gal is high or if theexpression level of the senescence markers Ki67 and/or Rb is low, thensaid tumor cell has high cannibal activity and undergo senescence, whatis of good prognosis value for the patient.

In a preferred embodiment, the said method comprises the step ofmeasuring the expression levels of p53 or of Δ133TP53, or the expressionlevel or the activity of the purinergic P2Y2 receptor, and theexpression level of a senescence marker such as p21^(WAF1), Ki67 p16,Rb, or SA-β-Gal, or of any other senescence marker as described above,in tumor cells isolated from the patient.

In a more preferred embodiment, the said method comprises the step ofmeasuring:

-   -   the expression levels of p53 or of Δ133TP53, and    -   the expression level or the activity of the purinergic P2Y2        receptor, and    -   the expression level of a senescence marker such as p21^(WAF1),        Ki67, p16, Rb, or SA-β-Gal, or of any other senescence marker as        described above, in tumor cells isolated from the patient.

In another aspect, the present invention also relates to an in vitromethod of determining the susceptibility of tumor cells to ananti-cancer treatment, comprising the step of detecting concomitantlythe cannibal and senescent behavior of the said cells by means of themethod of the invention, as defined above. As a matter of fact, if thesaid tumor cells exhibit simultaneously high cannibal and senescentactivities after treatment, then the said tumor cells will be sensitiveto the said anti-cancer treatment. In contrast thereto, if the saidtumor cells have high cannibal activity but low or no senescent activityafter treatment, then the said tumor cells will be resistant to the saidanti-cancer treatment.

By “concomitantly” or “simultaneously”, it is herein meant that thetarget processes occur in the cells from the same tumor. In particular,by using these terms, it is herein meant that the detection is performedin cells issued from the same tumor or patient, for example, from twotumor samples obtained from the same patient. As used herein, theseterms do not mean that the measuring steps should be performed at thevery same time, but rather on cell samples obtained from the samepatient, preferably from the same tumor.

Said anti-cancer treatment is preferably a radiotherapeutic treatment, achemotherapeutic treatment, an immunotherapeutic treatment, or acombination of same.

In these prognosis methods, the said tumor cells express normal levelsor high levels of non-imitated p53.

In these prognosis methods, the said cancer is preferably selected inthe group consisting of: melanoma, spitz tumors, prostate cancer, coloncarcinoma, and liver carcinoma, breast cancer, lung cancer, cervicalcancer, and bone cancer.

In another aspect, the present invention relates to treating methodscomprising the steps of:

-   -   Determining the susceptibility of tumor cells to a defined        anti-cancer treatment, by means of the prognosis method of the        invention, and, if said tumor cells exhibit simultaneously high        cannibal and senescent activities after treatment, then        maintaining the said anti-cancer treatment.    -   Whereas, if said tumor cells have high cannibal activity but low        or no senescent activity after treatment, then another        anti-cancer treatment is to be administered.

For example, if the patient is treated with a defined chemotherapeutictreatment, determining the susceptibility of tumor cells to saidtreatment by means of the method of the invention will enable theskilled person to decide if the said chemotherapy is required or if itwould be advantageous to replace—or to combine—it with radiotherapy orwith another chemotherapeutic treatment.

In a particular aspect, the present invention relates to an in vitromethod of determining the resistance of tumor cells to an anti-cancertreatment, comprising the step of detecting the cannibal and senescentbehavior of the said cells by means of the method of the invention, asdefined above. As a matter of fact, if the said tumor cells have highcannibal and senescent activities after treatment, then the said tumorcells will be sensitive to the said treatment. In contrast thereto, ifthe said cells have high cannibal activity but low or no senescentactivity, then the said tumor cells will be resistant to the saidanti-cancer treatment.

Said anti-cancer treatment is preferably a radiotherapeutic treatment, achemotherapeutic treatment, an immunotherapeutic treatment, or acombination of same.

In this particular aspect, the said tumor cells preferably expressmutated p53. These tumor cells are more preferably Triple NegativeBreast Cancer (TNBC).

For this particular aspect, the present invention finally relates totreating methods comprising the steps of:

-   -   Determining the susceptibility of tumor cells to a defined        anti-cancer e by means of the prognosis method of the invention,        and, if said tumor cells exhibit simultaneously high cannibal        and senescent activities after treatment, then maintaining the        said anti-cancer treatment.    -   Whereas, if said tumor cells have high cannibal activity but low        or no senescent activity after treatment, then another        anti-cancer treatment is to be administered.

For example, if the patient is treated with a defined chemotherapeutictreatment determining the susceptibility of tumor cells to saidtreatment by means of the method of the invention will enable theskilled person to decide if the said chemotherapy required or if itwould be advantageous to replace—or to combine—it with radiotherapy orwith another chemotherapeutic treatment.

In a preferred embodiment, all the prognosis methods of the inventioncomprises a step of detecting the cannibalism and/or senescence behaviorof the tumor cells on paraffin-embedded tumor tissue samples byimmunohistochemistry.

EXAMPLES

Cells, Antibodies and Reagents.

Colorectal carcinoma. (Tp53^(−/−) and Tp53^(−/−)) HCT116 cells werecultured in McCoy's 5A medium, Non small cell lung carcinoma H1299(p53^(−/−)), H1975, H1650, cervix adenocarcinoma HeLa cells,ostocarcinoma U20S cells and human fibroblasts WI38 were cultured inDulbecco's modified Eagle's medium (DMEM). Colorectal adenocarcinomaH1975 and H1650 cells and primary ductal carcinoma HCC1937 (p53^(−/−))cells were in RPMI-1640 Medium. All medium were supplemented 10% FCS, 2mM L-glutamine and 100 UI/ml penicillin/streptomycin (invitrogen). Headand neck squamous cell carcinoma SQ20B (p53^(R273H)) cells andpancreatic PANC813 cells were cultured as previously described. Allmedium were supplemented 10% FCS, 2 mM L-glutamine and 100 UI/mlpenicillin/streptomycin (Invitrogen). Human primary keratinocytes werecultured using KGM-Gold™ BulletKit™ Kit (Lonza, 00192060). Antibodiesfor detection of ATM, ATR, BRCA-1, BRCA-2, β-catenin, cleaved caspase-3(Asp175), FOXO-1, FOXO-3, FOXO-4 LKB1, p15INKb, p21^(WAF1), 53BP1, MLC2,MLC2S19*, PAR-4, PTEN, Rb, TP73 were obtained from Cell SignalingTechnology. Beclin-1, LAMP2, and TP53 (clone DO1) were from Santa Cruz.Antibodies against γ-H2AX and GAPDH were purchased from Millipore.Antibody against P2Y2 and TP53 (clone CM1) were respectively fromAlomone laboratories and Leica. Constructs containing TP53^(WT),TP53^(NES−), TP53^(NLS−), TP53^(R175H), TP53^(R273H) or EGFP and controlwere previously published (Tasdemir et al. 2008). Plasmids containingTP53β, TP53γ, Δ40TP53 and Δ133TP53 were obtained from Jean-ChristopheBourdon (University of Dundee UK).

RNA Interference.

Small interfering RNAs (siRNAs) specific for ATM (siRNA-1:5′UGAAGUCCAUUGCUAAUCA3′ and siRNA-2: 5′AACAUACUACUCAAAGACA3′), ATR(siRNA-1: 5′CCUCCGUGAUGUUGCUUGA3′ and siRNA-2: 5′GCCAAGACAAAUUCUGUGU3′), BRCA1 (siRNA-1: 5′ GCAACCUGUCUCCACAAAG3′ andsiRNA-2: 5′UGCCAAAGUAGCUAAUGUA3′), BRCA2 siRNA-1:5′CUGAGCAAGCCUCAGUCAA3′ and siRNA-2: 5′caacaauuacgaaccaaac3′), LKB1(siRNA-1: 5′GGACUGACGUGUAGAACAA3′ and siRNA-2: 5′ GUCCUUACGGCAAGGUGAA3′), p15INK4b (siRNA-1: 5CUCAGUGCAAACGCCUAGA3′ and siRNA-2:5′AACUCAGUGCAAACGCCUAGA3′), TP53 (siRNA-1: 5′GACUCCAGUGGUAAUCUAC3′ andsiRNA-2: 5′GCAUGAACCGGAGGCCCAU3′), TP63 siRNA-1:5′CCAUGAGGUGAGCCgUGAAu3′ and siRNA-2: 5′AGCAGCAAGUUUCGGACAG3′), TP73(siRNA-1: 5′CCACGAGCUCGGGAGGGAC3′ and siRNA-2: 5′ACGUCCAUGCUGGAAUCCG3′),Par-4 (siRNA-1: 5′GUGGGUUCCCUAGAUAUAA3′ and siRNA2:5′CAGCCGUUUGAAUAUAUUU3′), PTEN (siRNA-1: 5′GUCAGAGGCGCUAUGUGUA3′ andsiRNA-2: 5′CACCACAGUCUAGAACUUAU3′), Rb (siRNA-1: 5′GAAAGGACAUGUGAACUUA3′and siRNA-2: 5′CGAAAUCAGUGUCCAUAAA3′), p21WAF1 (siRNA-1:5′CUUCGACUUUGUCACCGAG3′ and siRNA-2: 5′CAGUUUGUGUGUCUUAAUUAU3′),Beclin-1 (siRNA-1:5′GAUUGAAGACACAGGAGGC3′ and siRNA-2:5′CCACUCUGUGAGGAAUGCACAGAUA3′), FOXO-1 (siRNA-1: 5′GCCCUGGCUCUCACAGCAA3′and siRNA-2: 5′CCGCGCAAGAGCAGCUCGU3′), FOXO-3 (siRNA-1: 5′GGGCGACAGCAACAGCUCU3′ and siRNA-2: GGAUGACGUCCAGGAUGAU3′), FOXO-4(siRNA-1: 5′CCCGACCAGAGAUCGCUAA3′ and siRNA-2: 5′GGACAAGGGUGACAGCAAC3′),Δ133TP53 (siRNA-1: 5′UGUUCACUUGUGCCCUGACUUUCAA3′ and siRNA-2:5′CUUGUGCCCUGACUUUCAA3′), P2Y2 (siRNA-1: 5′ GGAUAGAAGAUGUGUUGGG3′ andsiRNA-2: 5′GGCUGUAACUUAUACUAAA3′) and control siRNA(5′-GCCGGUAUGCCGGUUAAGU3′) were transfected 48 hours before cocultureusing Oligofectamine (invitrogen), according to manufacturer'sinstructions.

Pharmacological Inhibitions.

After inactivation of TP53 or Δ133TP53, primary human fibroblasts,primary human keratinocytes, colorectal carcinoma Tp53^(+/+) HCT116cells and pancreatic PANC813 cells were incubated during 24 hours inpresence or in absence of indicated concentrations of thepharmacological inhibitor of purinergic P2Y2 receptor Kaempferol(Sigma), the pharmacological inhibitor of ROCK Y27632 (Sigma) or thepan-caspase inhibitor zVAD-fmk (100 uM, BD Biosciences), ATP, UTP andsoluble apyrase used in indicated experiments were obtained from Sigma.

Immunoblots and Immunofluorescence.

Total cellular proteins were extracted in 250 mM NaCl-containing lysisbuffer (250 mM NaCl, 0.1% NP40, 5 mM EDTA 10 mM Na₃VO₄, 10 mM NaF, 5 mMDTT, 3 mM Na₄P₂O₇ and the protease inhibitor cocktail (EDTA-freeprotease inhibitor tablets, Roche). Protein extracts (50 μg) were run on4-12% SDS-PAGE and transferred at 4° C. onto a nitrocellulose membrane.After blocking, membranes were incubated with primary antibody at roomtemperature overnight. Horse radish peroxidase-conjugated goatanti-mouse or anti-rabbit (Southern Biotechnology) antibodies were thenincubated during 1 h and revealed with the enhanced ECL detectionsystem. After 48 hours of transfection with specific indicated siRNA,cells were stained with 10 μM of 5-chloromethylfluorescein diacetate(Cell Tracker Green CMFDA, Invitrogen) or with 10 μM of5-(and-6)-(((4-Chloromethyl)Benzoyl)Amino)Tetramethylrhodamine (CMTMR,Invitrogen) and cocultured for indicated time. Time-lapse microscopy wasperformed on 35 mm on cover glass bottom dishes coated with poly-hema.Fluorescence and differential interference contrast (DIC) or phasecontrast images were obtained every 5 min. For specific subcellularstainings of cannibal cells, cells were fixed in 4%paraformaldehyde/phosphate-buffered saline (PBS) for 30 minutes,permeabilized in 0.1% SDS in PBS and incubated with PCS for 20 minutes,as previously described (Seror et al., 2011). Antisera were used forimmunodetection in PBS containing 1 mg/ml BSA and revealed with goatanti-rabbit IgG conjugated to Alexa 546 fluorochromes and with rabbitanti-mouse IgG conjuguated to Alexa 647 fluorochromes from Invitrogen.Cells were counterstained with Hoechst 33342 (Invitrogen) and analyzedby fluorescent confocal microscopy on a Zeiss LSM510 using a63×objective.

Electron Microscopy.

After co-culturing of cell for 24 h, cells were fixed in 1.6%glutaraldehyde in 0.1 M Sorensen phosphate buffer (pH, 7.3) for 1 h at4° C. cells were washed one time with PBS and were refixed in aqueous 2%osmium tetroxide and finally embedded in Epon® epoxy resin, untilimaging. Examination is performed at 80 kV under a transmission electronmicroscopy, on ultrathin sections (80 nm) stained with 0.1% lead citrateand 10% uranyl acetate.

Measurement of Extracellular ATP.

ATP release was determined using the Enliten ATP assay system (Promega)as described by the manufacturer. The luminescence was measured byintegration over a 3-s time interval using the luminometer FluostarOPTIMA (BMG Labtech).

Cell Proliferation Assay.

Cancer cell lines (HCT116 WT, HCT116 Tp 53^(−/−, PANC)813) that stablyexpressed green or red fluorescent proteins in their nuclei (GFP-histoneH2B and RFP-histone H2B fusion proteins) were generated and werecocultured during 24 hours, optionally after depletion of Δ133TP53 toinduce cell-in-cell internalization. Cell-in-cell structures were seededin microtiter plates (with one single structure per well) by cellsorting using BD Influx™ cell sorter (Becton Dickinson). Sortedpopulations (single cell and cannibal cell) were validated underfluorescent microscope. Then, single cells and cannibal cells werecultured for seven days. During seven days, number of structure (singlecell or cannibal cell) were quantified daily.

Detection of Senescence Associated β Galactosidase.

After co-culturing of 48 h, cells were fixed and stained using thesenescence β-galactosidase staining kit (Cell signaling technologies) todetect senescence associated β galactosidase (SA-βGal) activity aspreviously described (Matsuura et al., 2007).

Cell Cycle Analysis.

To analyze cannibal cell proliferation, depleted cells were co-culturedfor 24 h and then incubated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU)for 1 h and stained using the Click-iT™ Edu Imaging Kit from Invitrogen.The cells were also counterstained with an antibody against β cateninand with Hoechst 33342 to identify cannibal cells and the nuclei werecounterstained with Hoechst 33342 (Invitrogen).

Senescence Induction.

Oncogene-induced senescence (OIS) was performed by retroviral-mediatedinfection of primary human diploid fibroblasts (HDF) strain WI38(population doubling 13) using pBABE-RAS^(V12) and Phoenix packagingcells. Twenty four hours post-infection, cells were pharmacologicallyselected with 4 μg/ml puromycin (pBABE) for 2 days. Day 0 is consideredwhen all non-infected cells were dead after pharmacological selection.Replicative senescent cells were produced by serial passaging of HDFs atnormoxic oxygen conditions of 3% until replicative exhaustion. Cellpopulations were considered senescent when less than onepopulation-doubling was completed per two weeks, EdU positivity was lessthan 1% and SA-b Gal positivity was greater than 70-80%.

Senescence Analysis.

Senescence was assessed using several assays. For growth curves, cellswere plated in triplicates at 2.0×10⁴ per well in 12-well plates.Relative cell numbers were estimated at various time-points using acrystal violet incorporation assay and population doublings (PD) werecalculated using the following equation: n=(log10^(F) log10^(I))×3.32(with n=PD, F=number of cells at the end of one passage, 1=number ofcells that were seeded at the beginning of one passage). For life-spanstudies, cells were sub-cultured when 70-80% confluent at 2×104/cm2.Proliferative capacity was assessed by labeling cells for 24 hours EdU.These cells were also co-stained for SA-β Gal.

Tumor patient selection. Breast cancer patients were treated with 3cycles of anthracycline and 3 cycles of docetaxel followed by surgery.As previously published (Chevallier et al, 1990), “Responders” exhibiteda complete pathological response with only few residual cancer cells(Chevallier score ≤1). “Non Reponders” (Chevallier score ≥2) exhibitedinvasive primary tumors or lymph node metastases.

Histology and immunochemistry. Samples from recovered tumors, mammarygland tissues and human breast adenocarcinoma were fixed with 4% PFA for4 h and then embedded into paraffin. Sections of 10 μm were fixed andstained with haematoxylin and eosin (HE) according to standardprotocols. For b-catenin Lamp2 and p21^(WAF1) immunochemistry antibodiesused are described in “Cells, antibodies and reagents” section.

Statistical analysis. To determine statistical significance, Student'st-test was used for calculation of P values.

Results

Identification of tumor suppressive protein p53 as a repressor ofcellular cannibalism. To identify the molecular basis of cancer-relatedcellular cannibalism, we determined the impact of the inactivation of 16tumor suppressor proteins on cell internalization, the first step ofcellular cannibalism. Among several cancer cell lines that spontaneouslymanifest cellular cannibalism (FIG. 6A), we chose the colon carcinomaHCT116 cell line to determine cell internalization after inactivation oftumor suppressor proteins involved in cell cycle regulation (LKB1, PTEN,Rb and p15INK4b), DNA repair (BRCA-1 and BRCA-2), DNA damage responses(ATM and ATR) and cell death regulation (FOXO-1, FOXO-2, FOXO-3,Beclin-1, Par-4, TP53, TP63 and TP73). After tumor suppressor knockdown,half of the cells were labeled with 5-chloromethylfluorescein diacetate(CMFDA, green fluorescence) and the other half with5-(and-6)-(((4-Chloromethyl)Benzoyl)Amino)Tetramethylrhodarnine (CMTMR,red fluorescence), mixed together, and cultured for 24 hours (FIG. 1Aand FIGS. 6B-Q). Subsequent, confocal microscopy revealed that depletionof TP53 resulted in a significantly enhanced frequency of cell-in-cellstructures (FIGS. 1B and 1C). Cell internalization was confirmed byimmunostaining of membrane-bound catenin revealing that one engulfingcell (“cannibal”) can internalize more than one—up to six—cells(“targets”) (FIG. 1D and FIG. 7A), Neither CMFDA nor CMTMR did affectthe frequency of cell-in-cell structures (not shown) or the propensityof cells to act as cannibals or targets (FIG. 1E), although theyprovided a better resolution for the detection of cellular cannibalismthan classical immunohistochemistry staining (FIG. 7B). Time-lapsemicroscopy of CMFDA/CMTMR-labelled, TP53-depleted cells demonstratedthat cellular internalization occurred without morphological signs ofapoptosis (such as membrane blebbing and formation of apoptotic bodies)(FIG. 7C), in line with an absent activation of caspase-3 (FIG. 7D). Theinternalization step was not related in its timing to symmetric orasymmetric mitoses of p53 depleted cells (FIG. 7E). Pharmacologicalinactivation of TP53 with cyclic by pifithrin-α (FIG. 1F,G),siRNA-mediated depletion of TP53 (FIGS. 1F-I), or knockout of the Tp53gene by homologous recombination (FIG. 1F,G) induced cell-in-cellstructures in HCT116 cells (FIG. 1F,G), as well as in human primaryfibroblasts (FIG. 1H,I). Irrespective of the method of TP53inactivation, inhibition of the serine threonine kinase ROCK by Y26732(Overholtzer et al., 2007), prevented the generation of cell-in-cellstructures (FIGS. 1F-I and FIGS. 2F-I), which however were not affectedby the pan-caspase inhibitor Z-VAD-fmk (FIGS. 1F-I). Overall, ourresults identified a novel role for TP53 as a repressor of cellularcannibalism.

Δ133p53 is required for cellular cannibalism repression. Next, we used abattery of p53 mutants and isoforms to explore the mechanisms thatgovern the TP53-mediated regulation of cellular cannibalism. Wild type,full-length TP53, as well as a purely nuclear TP53 mutant (which lacksthe nuclear export sequence, NES) were able to repress the formation ofcell-in-cell structures, while a purely cytoplasmic TP53 mutant (whichlacks a nuclear localization sequence, NLS) or two mutants that lack thetransactivation activity of TP53 (due to frequent, cancer-associatedmutations: R175H or R273H were unable to repress the cannibalisticactivity (FIG. 2A-D). All TP53 splice variants that differ in the startcodon and hence in their N-terminus (TP53β, TP53γ, Δ40TP53 andΔ133TP53), including the shortest version (Δ133TP53), which lacks theamino-terminal trans ovation and prolin-rich domains, were able toinhibit cannibalism (FIG. 2E,F). More importantly, selective depletionof Δ133TP53 with two distinct siRNAs that do not affect any otherisoforms (FIG. 2G, (Fujita et al., 2009)) could induce cannibalism inseveral distinct cancer cell lines (HCT116, PANC) and primary humanfibroblasts (FIG. 2H-L, FIG. 8A,B), Knockdown of all TP53 isoforms orthat of Δ133TP53 alone similarly induced cannibalism.

In order to determine cellular consequences of Δ133p53 depletion, siRNAmediated knock down of Δ133p53 isoform from human colon HCT116 cells(FIG. 2G-I), human pancreatic PANC813 cells (FIG. 2J) or from humannormal WI39 fibroblasts (FIG. 2K,L) was performed during 48 hours. Then,control and depleted cells were labeled with either CMFDA or CMTMR,mixed together and cultured during 24 hours. Confocal microscopyanalysis of these experiments revealed that reduction of Δ133TP53expression induced ROCK-dependent and caspase-independent cannibalism incancer cells (HCT116 and PANC) (FIG. 2I,J), but also in normal humanfibroblasts (WI39) (FIG. 2L). Next, we examined whether TP53 andΔ133TP53 act on cannibal cells or target cells. CMFDA-labeled (green) WTHCT116 cells (which express TP53) were co-cultured with CMTMR-labeled(red) cells that were manipulated for p53 expression: either WT cellstransfected with s control siRNA, WT cells depleted from TP53 orΔ133IP53 only, or Tp53^(−/−) cells. In this system, the absence of TP53or Δ133TP53 alone enhanced the frequency of green-in-red cellstructures, but not that of red-in-green figures (FIG. 2M). Hence, TP53and Δ133TP53 act on the side of cannibal cells, not that of targetcells, meaning that they increase the activity of cellularinternalization on the side of the engulfing (not the engulfed) cell.

Extracellular ATP and P2Y2 purinergic receptors contribute tocannibalism. Stressed and dying cells can expose ‘come get me’ and ‘eatme’ signals or lose ‘don't eat me’ signals, thus facilitating theirengulfment by neighboring cells (Grimsley and Ravichandran, 2003). Thenucleotide ATP is released by stressed or dying cells and constitutes apotent chemotactic signal (Ravichandran, 2011) that can contribute tocell-to-cell contacts and fusions (Seror et al., 2011; Trautmann, 2009).Accordingly, tp53^(−/−) cells (FIG. 3A), as well as irradiated or CDDPtreated cells (FIG. 11B) and TP53 or Δ133TP53 depleted cells (FIG. 11C)released ATP shortly after treatments (FIG. 11B) or during coculture(FIG. 3C and FIG. 11C). These processes require ROCK signaling pathway(FIG. 11B-D). Importantly, addition of recombinant apyrase, an enzymethat degrades ATP and ADP to AMP, reduced cellular internalizationinduced by the depletion of TP53 or Δ133TP53 in HCT116 (FIG. 3B).Conversely, supplementation with ATP or UTP strongly enhanced theROCK-dependent generation of cell-in-cell structures (FIG. 3C,D),confirming that these nucleotides can stimulate cellular cannibalism.Accordingly, the deletion or depletion of TP53 or Δ133TP53,respectively, enhanced the expression of the P2Y2 purinergic receptors(FIG. 3E,F), which are known to mediate ATP-dependent engulfment signals(Chekeni et al., 2010). In addition, 3% oxygen treated HDFs (FIG. 11F),senescent (FIG. 11G) and IR treated HEKns (FIG. 11H) and CDDP or IRtreated cells (FIG. 11I) enhanced the expression of the P2Y2 purinergicreceptors. Knockdown of this particular purinergic receptor by twonon-overlapping siRNAs reduced cellular cannibalism resulting from TP53inhibition or depletion in HCT116 cells (FIG. 3G), as well as in PANC813cells (FIG. 3H). Pharmacological inhibition of the upregulatedpurinergic P2Y2 receptor also reduced the cannibalism of human primaryfibroblasts depleted from TP53 or ΔA133TP53 (FIG. 3I-K) or stressedafter replication (FIG. 11J). Considering that p53 acts on host cells torepress cellular cannibalism (FIG. 2M), we decided to determine whetherp53 inactivation controls P2Y2 activity on target cell side or/and hostcell side. We revealed that the depletion of P2Y2 receptors on hostcells reduced the ROCK-mediated phosphorylation of light chain 2 ofmyosine (data not shown) and cellular cannibalism (FIG. 11K)demonstrating that the ATP-driven activation of P2Y2 receptors on hostcells favors cellular cannibalism.

Cellular invasion triggers senescence of cannibal cells. Confirmingprior reports on entosis that describe the fatal destiny of engulfedcells (Overholtzer et al., Cell 2007), internalized cells were targetedto LAMP2^(H) lysosomal compartments for destruction, irrespective of thecell type that was analyzed (HCT116 or PANC813) after TP53 deletion orΔ133TP53 depletion (FIG. 9A-D), and cellular internalization wasfollowed by nuclear degradation in an acidic compartment (FIG. 9E-G). Incontrast cannibal cells conserved their viability. To investigate theirlong-term fate, we generated isogenic pairs of cancer cell linesHCT116WT , HCT116 Tp53^(−/−), PANC813) that stably expressed green orred fluorescent proteins in their nuclei (GFP-histone H2B andRFP-histone H12B fusion proteins). Mixtures of such cells werecocultured, optionally after depletion of Δ133TP53 to induce entosis,and cell-in-cell structures were seeded in microtiter plates (with onesingle structure per well) and cultured for several days (FIG. 4A andFIG. 10A,B). In contrast to single control cells, cell-in-cellstructured formed from CT116 WT or HCT116 Tp53^(−/−) cells that weredepleted from Δ133TP53 were unable to proliferate (and hence to increasetheir cell number), and demonstrated an increase in cell size andnuclear size, corresponding to an increase in ploidy (FIG. 4B,C). Thiscorrelated with reduced incorporation of the DNA precursor5-ethynyl-2′-deoxyuridine (EdU), a thymidine analogue (FIG. 4C,D),increased staining of the nuclei from cannibal (but not target) cellswith antibody recognizing phosphorylated histone H2AX (γH2AX DNA damagefoci) (FIG. 4E,F) or p53BP1 (which also labels DNA damage foci (Lukas etal., 2011)) (FIG. 4E,F), and cytoplasmic senescence associatedβ-galactosidase (SA-β-Gal) activity (FIG. 4G,H), as well as expressionof the cycline-dependent kinase inhibitory protein p21^(WAF1) (FIG.4I-K), siRNA-mediated depletion of p21^(WAF1) reduced sings ofsenescence including SA-β-Gal staining (FIG. 4L-N) and allowed the cellsto resume EdU incorporation (data not shown). According to the wordentosis that defined a non-apoptotic cell death mechanism occuring aftercell internalization, we termed this new modality of senescenceinduction, entescence.

Altogether, these results provide evidence for a link between Δ133p53 orp53 depletion, senescence and cellular cannibalism.

Senescence favors cannibalism. Oncogenic stress-induced senescencetriggered by transducing primary human WI38 fibroblasts with Ras^(V12)(FIG. 5A-C) led to an increase in P2Y2 expression (FIG. 5A), parallelingan increase in p16 and a loss of Rb expression, two phenomena that areknown to be associated with senescence (Lowe et al., Nature 2004). Inaddition, Ras^(V12) mediated retroviral transduction resulted in anincrease in the frequency of cannibalistic events, allowing theRas^(V12)-expressing fibroblasts to engulf HCT116 cells or normalnon-senescent fibroblasts (FIG. 5B,C), through a process that dependedon ROCK but not on caspases (FIG. 5C), Replicative stress induced byreplicative exhaustion of HDFs at 3% normoxic oxygen conditions ofupregulated P2Y2 (FIG. 5D) and stimulated cannibalism (FIG. 5E).Inhibition of P2Y2 receptors reduced the frequency of cannibalisticevents induced by Ras^(V12) (FIG. 5F). In addition, replicativesenescence of human primary epidermal keratinocyte (HEKn) (Rivetti diVal Cervo et al., PNAS 2012) led to the induction of P2Y2 protein (FIG.5G) and elevated cannibalism (FIG. 5H) requiring ROCK and P2Y2 (FIG.5I). Prevention of cannibalism with inhibitors of ROCK or P2Y2 alsoreduced the frequency of senescent, SA-β-Gal-positive cells accumulatingin aging cultures (FIG. 5J), These results support the ability ofsenescent cells to manifest a cannibalistic behavior and to engulfneighboring cells.

Entescence Contributes to Cellular Senescence Through P2Y2 Activation.

To further characterize the role of cellular cannibalism duringsenescence induction, we evaluated the impact of purinergic P2Y2receptor activity on entescence and on cell autonomous senescence andobserved that the pharmacological inhibition of purinergic P2Y2 receptorwith kaempferol strongly reduced senescence observed during replicativestress of HEKns (FIG. 12A) or after treatment of HCT116 cells with IR orwith MTX (FIG. 12B) as revealed by the reduction of the frequency ofsenescent, SA-β-Gal-positive cannibal cells (FIGS. 12A,B). Then, weevaluated the contribution of P2Y2 signaling pathways on p53 depletioninduced entescence and revealed that P2Y2 depletion reducedsignificantly entescence detected after p53 deletion or depletion (FIG.12C). These results were also confirmed after the depletion of thesenescence regulator Δ133p53. As we previously described the depletionof Δ133p53 isoform from human colon HCT116 cells triggers cellularcannibalism (FIGS. 3I,J). Time lapse imaging of Δ133p53 depleted HCT116cells that stably expressed GFP-histone H2B and RFP-histone H2B fusionproteins highlighted that once internalized target cells are degradedwithin less than one hour and then, cannibal cells increased their sizeand nuclear size (FIG. 12D). This process is associated with anincreased staining of the nuclei from cannibal cells with antibodiesrecognizing γH2AX DNA damage foci (FIGS. 12E,G) or p21 (FIGS. 12E,H),and the induction of SA-β-Gal activity (FIGS. 12F,J) demonstrating thatΔ133p53 depleted HCT116 cells undergo entescence. Depletion of p21 bytwo siRNA revealed reduced entescence (FIG. 4N), but also entescenceassociated cannibalism (FIG. 4O) highlighting that senescent cells andentescent cells are both able to internalize neighboring cells. We alsodemonstrated that P2Y2 depletion reduced specifically the frequency ofcannibal cells showing for γH2AX positive nuclear foci (FIG. 5G), p21expression (FIG. 5H) and SA-β-Gal activity (FIG. 5I) revealing that thepurinergic P2Y2 receptor modulate cellular senescence by controlling theengulfment of neighboring cells. Taken together, these data demonstratedthat entescence contributes to the establishment of cellular senescence.

Detection of Entescence In Vivo

Despite cell-in-cell cytological feature of cellular cannibalism hasbeen detected in numerous human cancer, role of cellular cannibalismduring oncogenesis was only recently studied (Overholtzer et al., Naturereviews Molecular cell biology 2008; Cano et al., EMBO Mol. Med. 2012)and depending on tumor microenvironment, could either contribute totumor suppression through entosis induction {Overholtzer et al., Cell2007; Cano et al., EMBO Mol. Med. 2012), but could also increase genomicinstability of cannibal cells by modulating cytokinesis (Krajcovic etal., Nat Cell Biol. 2011), To validate our vitro based hypothesisdemonstrating that cellular cannibalism is associated with senescence,we decided to determine whether human Spitz tumors that contain manysenescent cells also show presence of cannibal cells. We analyzed humanSpitz tumors and human aggressive melanomas using HE andimmunofluorescence stainings and detected an increase of cannibal cellsin Spitz tumor biopsies, as compared to aggressive melanoma (data notshown). In comparison to malignant melanomas which are known to bedeleted or mutated for p16^(INK4a) (Kamb et al, Nat Genet 1994), themajority of these begnin tumors contain senescent cells highlightingthat cellular cannibalism is also associated with senescence in vivo. Inaddition, these results also suggest that induction of cellularcannibalism would determine the long-term survival of patients andanimals to chemotherapy Schmitt et al. Nature reviews Cancer, 2003; Loweet al., Nature 2004). To confirm this hypothesis, we determined whethercannibal cells detected on human breast adenocarcinoma could undergosenescence after neo-adjuvant treatment. First, we analyzed normal humanbreast and primary breast carcinoma biopsies using the previousexperimental strategy and found that all biopsies revealed feature ofcellular cannibalism (FIG. 13A and FIG. 13B), The frequency of cellengulfment was 10 fold higher in primary breast carcinomas (mean+/−SD=,n=30) than in normal tissues (mean+/−SD=, n=10) (FIG. 13C). To determinewhether cellular cannibalism correlates with disease progression, weanalyzed 10 biopsies of patients diagnosed with a grade 1, with a grade2 or a grade 3 of breast carcinomas and observed that the frequency ofcannibal cells significantly increased with the histological tumor grade(FIG. 13D). To precise the impact of cellular cannibalism on breastcancer progression, we wondered to know whether of neo-adjuvanttreatments could increase cellular cannibalism and senescence ofcannibal cells. We examined 30 biopsies of untreated breast carcinomasand 15 neo-adjuvant treated breast carcinomas for cell engulfment and wefound no changes in the frequency of cannibal cells, but we observedcannibal cells with an increase in cell and nuclear sizes inneo-adjuvant treated tumor biopsies as compared to untreated (FIG. 13Ea-c). Then, we stained sections from 30 untreated tumors and 15 treatedtumors with for β-catenin and p21^(WAF1) antibodies (FIG. 13F) andobserved in half of neo-adjuvant treated tumors that single and cannibalcells are positive for p21^(WAF1+) (FIG. 13G), confirming our previousresults on Spitz tumors that cellular cannibalism and senescence are twointerlinked processes that can also be detected in patients afterneo-adjuvant treatments in vivo. Considering that breast carcinomasrepresent a heterogeneous group of tumors with different morphologicaland biological features, behavior and response to therapy, we comparedthe impact of cellular cannibalism and senescence on tumor and diseaseprogressions between to distinct types of breast tumor that were treatedwith neo-adjuvant treatment. First, we determined the frequency ofcellular cannibalism on breast tumor biopsies obtained from 26 patientswith locally advanced breast cancers and observed that the efficiency ofneo-adjuvant chemotherapy is associated with induction of cellularcannibalism (FIG. 14H). Cannibal cells detected in responder patientsrevealed evidences of cellular senescence (such as increase of theircellular and nuclear sizes, expression of p21^(WAF1) and absence of Ki67staining). In contrast, biopsies from non-responder patients revealed astrong reduction of cellular cannibalism and did not present signs ofentescence or cellular cannibalism associated to senescence. Moreimportant, we found that the detection of cellular cannibalism isassociated with an increase of disease free and overall survivals oftreated patients (FIG. 14B and FIG. 14C) suggesting that the detectionof cellular cannibalism (and more precisely senescence of cannibalcells) on biopsies obtained from breast tumors strongly predict responseto nee-adjuvant treatment. Then, we determined whether the detection ofcellular cannibalism in triple negative breast cancer tumors (TNBC) thatare more aggressive tumors could also predict the efficiency ofneo-adjuvant treatment. We evaluated the frequency of cannibal cells on71 TNBC biopsies obtained after neo-adjuvant treatment and revealed thatthe detection of cellular cannibalism also predict the efficiency ofneo-adjuvant treatment on TNBC patients, but with great interest inthese tumors that harbor p53 mutation and don't undergo senescence, theaccumulation of cellular cannibalism is associated with the absence ofresponse to neo-adjuvant treatment (FIG. 14D) and a strong reduction ofdisease free survival and overall survival of treated patients (FIG. 14Eand FIG. 14F). These results confirmed those obtained in FIG. 15demonstrating that p53 mutations repress entescence and lead to theaccumulation of cannibal polyploidy cells that are able to divide andrestart proliferation.

Overall, these data obtained in vivo confirm that entescence or/andsenescence associated to cellular cannibalism also occur in patients andtheir detections could help for the determination of disease outcomesand for the prediction of chemo- and radiotherapy efficiency.

Discussion

Here, we show that pharmacological and genetic inhibitions of TP53 orΔ133TP53 initiate cellular cannibalism by inducing ATP release and P2Y2activation. After internalizing target cells, cannibal cells becomesenescent through p21^(WAF1) induction. Once senescent, cannibal cellscan also internalize other lived neighboring cells. These findingsprovide the first demonstration that cellular cannibalism may modulatetumor growth despite TP53 or Δ133TP53 expression is reduced.

Although sig ling pathways elicited by TP53 inactivation wereextensively investigated, we show that ATP is released into theextracellular milieu during cell interactions between TP53 deficientcells or between Δ133TP53 depleted cells (FIG. 3A and data not shown)and that this extracellular ATP is required for cell internalization(FIG. 3B-D). Release of ATP by cells occurs through multiple mechanismsthat may activate membrane channels eluding connexin and pannexinhemichannels) or induce exocytic or autophagic mechanisms Corriden andInset, 2010). Autophagy also known as “self-eating” is a cellularresponse to multiple stresses that TP53 was recently identified asregulator of autophagy. Deletion, depletion and inhibition of TP53 caninduce autophagy in human, mouse and nematode cells (Tasdemir et al.,2008), but autophagy is also required for ATP release from dying tumorcells during chemotherapy cells and contributes to immunogenicity. Inthis context, we postulated that release of ATP that we detected duringculture of TP53 cells (FIG. 3A) or Δ133TP53 depleted cells (data notshown) could be initiated by autophagy. Further characterization ofmechanisms that are required for ATP release will determine the role ofautophagy in cellular cannibalism. During entosis, autophagy machinerywas involved in the degradation of internalized cell (Florey et al., NatCell Biol. 2011; Florey and Overholtzer, Trends Cell Biol. 2012), butimpact of autophagy on cell-in-cell structure formation remains to beinvestigated.

Once release, extracellular ATP can exert a wide range of cellulareffects by activating plasma membrane bound receptors such as ionotropicpurinergic P2X receptors and metabolotropic PINT receptors (Burnstock,Cell Mol Life Sci. 2007). In this study, we revealed that TP53 or Δ33TP3knockdowns increase expression of heterotrimeric guanine nucleotidebinding pro (protein G) coupled P2Y2 receptors in normal and in cancer(FIG. 3). More important, P2Y2 depletion impairs cell engulfment andreduces drastically educes frequencies of cell-in-cell structuresdetected after TP53 or Δ133TP53 inactivation, demonstrating that P2Y2participates in the engulfment of neighboring cells by cannibal cells.Purinergic P2Y2 receptor contributes to actine cytoskeletonrearrangement that is required for cell interactions and cell fusion(Paoletti et al., PNAS 2012; Seror et al., 2011) and increases mobilityof cells through activation of Rho and formation of Rho dependent stressfibers (Bagchi et al., 2005; Chen et al., 2006; Liao et al., 2007; Liuet al., 2004). Indeed, depletion of TP53 changes the morphology and thepolarity of cells (Gadea et al., 2007; Gadea et al., 2002; WO et al.,2003), but also increases mobility in numerous cell types (includingkeratinocytes and colon cancer HCT116 cells) (Lefort et al., 2007;Sablina et al., 2003). Evidences that signaling pathways modulating cellmigration and chemotaxis are influenced by TP53 have been also produced(Gadea et al., 2007; Gadea et al., 2002; Guo and Zheng, 2004; Lefort etal., 2007; Roger et al., 2006; Xia and Land, 2007), demonstrating thatTP53 loss drives increased activity of Rho/ROCK axis to promote cellmobility and invasion during tumor progression. Additional experimentshave to be developed to determine the link between extracellular ATP andpurinergic P2Y2 activation, and Rho/ROCK dependent signaling pathwaysduring cellular cannibalism. Our results suggest that after TP53 orΔ133TP53 depletion, extracellular ATP and purinergic P2Y2 receptorsactivation may enhance mobility of cannibal cells and favor engulfmentof neighboring cells.

Cellular cannibalism was initially described as non apoptotic cell deaththat is provoked by loss of attachment to extracellular matrix and ledto destruction of internalized cells through lysosomal degradation. Thisatypical cell death modality was defined as mitosis and was proposed asa new tumor suppressor mechanism to control tumor growth and metastaticdissemination (Overholtzer et al., 2007). More recently, studies oncellular cannibalism demonstrated that internalizing cells (that wepreviously defined as cannibal cells) cause a cytokinesis failure ofcannibal cells by disrupting the formation of the contractile ringduring cannibal cell division. Hence, cellular cannibalism initiates anon-genetic mechanism of cytokinesis failure and increase genomicinstability by generating aneuploidy cells (Krajcovic et al., 2011;Krajcovic and Overholtzer, 2012). Here we demonstrated that cellengulfment induces hyperploidy and senescence of cannibal cells whenTP53 or Δ133TP3 is reduced. Senescence is a stable cell cycle arrestthat is induced at the end of the cellular lifespan or in response todifferent stresses (such as replicative or oxidative stresses, DNAdamage, chemotherapeutic drugs). No specific markers for senescence havebeen identified, but senescent status of cells is determined bydetecting an irreversible growth arrest, the expression of thecytoplasmic senescence associated β-galactosidase (SA-βGal), there-expression of cell cycle inhibitors (such as p16^(INK4a) orp21^(WAF1)), the secretion of numerous growth factors, cytokines andproteases; and by the presence of nuclear foci that contain DNA damageresponse (DDR) proteins or heterochromatin. We found that cannibal cellsshowed an irreversible growth arrest, expressed cytoplasmic senescenceassociated β-galactosidase (SA-β-Gal) activity and the cycline-dependentkinase inhibitory protein p21^(WAF1). We also detected DDR foci in thenuclei of cannibal cells demonstrating, that in absence of TP53 orΔ133TP53, cancer cells become senescent after cell engulfment. Althoughtranscriptional activity of TP53 is frequently required for theinduction of senescence, we unrevealed that senescence induction occursin absence of TP53 or its Δ133TP53. Our results were in accord withpreviously published reports demonstrating that cellular senescence maybe induced in melanoma in absence of TP53 (Ha et al., 2007; Ha et al.,2008) or by ARF independent TP53 signaling pathways (Lin et al., 2010).Recently, decrease of Δ133TP53 and overexpression of TP5313 participateto replicative senescence, but not oncogene induced senescence (Fujitaet al., 2009), In addition, we also detected on cannibal cells, DNAdamage responses (as revealed by nuclear foci containing γ-H2AX and53BP1) and p21^(WAF1) over-expression two cellular processes that arerequired for replicative or oncogenic senescence. Inhibition of p21expression reduced senescence of cannibal cells depleted for TP53 or forΔ133TP53, suggesting that p21^(WAF1) overexpression is induced throughTP53-dependent or independent mechanisms. Cellular senescence is acrucial barrier to tumor progression in vivo (Kuilman et al., 2010),Understanding molecular and cellular basis of senescence represent acrucial step to fight cancer and develop therapeutic strategies (such aspro-senescent therapy (Nardella et al., 2011)) to reduce tumor growthand metastatic dissemination, Here, we highlighted a new mechanism fortumor suppression that could be enhanced for therapeutic benefits whenTP signaling pathways are impaired.

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The invention claimed is:
 1. An in vitro method for detecting cannibalism behavior of a cell, comprising: measuring expression level of p53, expression level of a N-terminal isoform of p53 that lacks N-terminal transactivating domain, expression level of p53β, or expression level of p53γ in said cell, wherein measuring expression level comprises measuring mRNA level; and measuring activity of purinergic P2Y2 receptor (P2Y2R) in said cell by determining the phosphorylation of Pyk2.
 2. The method according to claim 1, wherein the N-terminal isoform of p53 that lacks N-terminal transactivating domain is Δ40TP53 or Δ133TP53.
 3. The method according to claim 1, further comprising measuring at least one senescence marker.
 4. The method according to claim 3, wherein the senescence marker is p21WAF1, Ki67, p16, Rb, or SA-β-Gal.
 5. The method according to claim 1, wherein said cell is a cancer cell.
 6. The method of claim 5, wherein said cell is a melanoma, spitz tumor, prostate cancer, colon carcinoma, liver carcinoma, breast cancer, lung cancer, cervical cancer, or a bone cancer cell.
 7. The method of claim 6, wherein said cell is a Triple Negative Breast Cancer cell.
 8. The method of claim 1, wherein the measuring steps are performed on a paraffin-embedded tumor tissue sample comprising the cell.
 9. The method according to claim 1, wherein measuring activity of P2Y2R in said cell comprises determining the phosphorylation of Pyk2 on tyrosine
 402. 10. An in vitro method for detecting cannibalism behavior of a cell, comprising: measuring expression level of p53, expression level of a N-terminal isoform of p53 that lacks N-terminal transactivating domain, expression level of p53β, or expression level of p53γ in said cell; and measuring activity of purinergic P2Y2 receptor (P2Y2R) in said cell by determining the phosphorylation of Pyk2, wherein said cell is a cancer cell.
 11. The method of claim 10, wherein said cell is a melanoma, spitz tumor, prostate cancer, colon carcinoma, liver carcinoma, breast cancer, lung cancer, cervical cancer, or a bone cancer cell.
 12. The method of claim 11, wherein said cell is a Triple Negative Breast Cancer cell.
 13. The method of claim 10, wherein the measuring steps are performed on a paraffin-embedded tumor tissue sample comprising the cell.
 14. The method according to claim 10, wherein measuring activity of P2Y2R in said cell comprises determining the phosphorylation of Pyk2 on tyrosine
 402. 15. The method according to claim 14, wherein measuring expression level comprises measuring mRNA level. 